Selection Method and Recombinant Microorganisms and uses Therefor

ABSTRACT

One or more genes in a biosynthesis pathway for a vitamin or other essential nutrient which is needed for the survival of a microorganism can be used as an effective selective marker to identify cells transformed with an exogenous nucleic acid. The microorganism does not naturally contain or express the one or more gene. This permits genetic manipulations to be performed. It permits lower cost fermentations to be performed. It permits production of the essential nutrient for subsequent commodity use.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application No.61/650,757 filed on 23 May 2012 which contents are incorporated byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to recombinant microorganisms, methods for theproduction of one or more products by fermentation, and selectionmethods.

BACKGROUND OF THE INVENTION

Selectable (or selective) markers and agents are important forgenetically modifying cells or microorganisms. They can be used toscreen for cells with introduced DNA: A selectable marker protects theorganism from the presence or absence of a selective agent that wouldnormally kill it or prevent its growth. Usually antibiotic resistancegenes are used as selectable markers, and the respective antibiotic asselective agent.

Use of selectable agents like antibiotics adds significant costs to aprocess like an industrial fermentation. Some antibiotics are also notor only poorly soluble in water and need to be dissolved in solvents.This adds further costs and potentially has a negative effect on themicrobial cells; for example, chloramphenicol and thiamphenicol need tobe dissolved in Ethanol or Dimethylformamide (DMF), respectively.

Some organisms are also naturally resistant to some antibiotics and sothey can not be used as selectable agents. For example, most antibioticsclasses are only active against either Gram-positive or Gram-negativebacteria as they target cell wall compounds and several Genera havenatural resistance against specific antibiotics (for example, Clostridiaagainst Chloramphenicol, as they posses chloramphenicolacetyltransferase genes that confer resistance and are also able toreduce the aryl-nitro-residue of the molecule to inactivate it (O'Brien& Morris, 1971, J Gen Microbiol, 67: 265-271)).

In addition, some antibiotic substances get inactivated or cannot beused under typical process conditions. For example, the low pH between4-5.5 used in several fermentation processes inactivates the macrolideerythromycin, while on the other hand it's analogue clarithromycin onlydissolves at extremely low pH below 2 (Mermelstein & Papoutsakis, 1993,FEMS Microbiol Lett, 113: 71-76).

Auxotrophic markers that can compensate for an inability to metabolisecertain amino acids, nucleotides, or sugars can also be used forselection. However, these also require the addition of compounds to themedia which are not otherwise needed, increasing expense.

Reporter genes have also been used to allow for selection of successfultransformants during processes for producing recombinant microorganisms;for example, genes encoding green fluorescent protein orbeta-galactosidase (lacZ). However, these can be toxic to the cells andthe products produced undesirable in commercial fermentation reactions.

It is an object of the invention to overcome one or more of thedisadvantages of the prior art, or to at least to provide the publicwith a useful choice.

SUMMARY OF THE INVENTION

The invention generally provides, inter alia, methods for the productionof one or more products by microbial fermentation of a substrate,genetically modified microorganisms of use in such methods, nucleicacids suitable for preparation of genetically modified microorganisms,and methods for the selection of certain microorganisms in a mixedpopulation of microorganisms or prevention of the growth of undesirablemicroorganisms.

In a first aspect, the invention provides an recombinant microorganismcomprising at least one exogenous nucleic acid adapted to express one ormore enzymes in one or more vitamin biosynthesis pathway, such that therecombinant microorganism can produce the one or more vitamin(s),wherein the recombinant microorganism is an anaerobe.

In one embodiment, the at least one exogenous nucleic acid encodes oneor more gene encoding one or more enzymes in the one or more vitaminbiosynthesis pathway.

In one embodiment, the one or more vitamin is needed for growth of themicroorganism. In one embodiment, the one or more vitamin is essentialfor growth of the microorganism.

In one embodiment, the at least one exogenous nucleic acid comprises oneor more gene which is lacking in a parental microorganism from which therecombinant microorganism is derived.

In one embodiment, the invention provides a recombinant microorganismcapable of producing one or more enzyme of one or more vitaminbiosynthesis pathway, the microorganism comprising one or more exogenousnucleic acid encoding the one or more enzyme, wherein the recombinantmicroorganism is derived from a parental microorganism that lacks one ormore nucleic acid encoding the one or more enzyme.

In one embodiment, the one or more vitamin is chosen from the groupcomprising thiamine, pathothenate, riboflavin, nicotinic acid,pyridoxine, biotin, folic acid, and cyanocobalamine. In one particularembodiment, the vitamin is thiamine (B1) and/or panthothenate (B5).

In one embodiment, the one or more of the enzymes is chosen from thegroup herein after described. In one particular embodiment, the one ormore enzymes is chosen from thiamine biosynthesis protein ThiC (EC4.1.99.17), 3-methyl-2-oxobutanoate hydroxymethyltransferase PanB (EC2.1.2.11), pantoate-beta-alanine ligase PanC (EC 6.3.2.1), and aspartate1-decarboxylase Pan D (EC 4.1.1.11). In one embodiment, the enzyme isThiC. In another embodiment, the one or more enzymes are PanB, PanC andPanD, in combination.

In one embodiment, the microorganism is selected from the groupcomprising In one particular embodiment, the microorganism is selectedfrom the group comprising Genera Clostridium, Eubacterium,Peptostreptococcus, Peptococcus, Actinomyces, Lactobacillus,Bifidobacterium, Propionibacterium, Bacteriodes, Fusobacterium,Campylobacter, or Veillonella.

In one embodiment, the microorganism is a carboxydotrophic acetogenicbacteria.

In one particular embodiment, the microorganism is selected from thegroup comprising Clostridium autoethanogenum, Clostridium ljungdahlii,Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei,Clostridium scatologenes, Clostridium aceticum, Clostridiumformicoaceticum, Clostridium magnum, Butyribacterium methylotrophicum,Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta,Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica,Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacterpfennigii, and Thermoanaerobacter kiuvi.

In one embodiment the microorganism is Clostridium autoethanogenum orClostridium ljungdahlii. In one particular embodiment, the microorganismis Clostridium autoethanogenum DSM23693. In another particularembodiment, the microorganism is Clostridium ljungdahlii DSM13528 (orATCC55383).

In one particular embodiment, the microorgism is chosen from Clostridiumautoethanogenum or Clostridium ljungdahlii, and the enzyme is ThiC. Inanother particular embodiment, the microorganism is Clostridiumautoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei orAcetobacterium woodii and the enzymes are PanB, PanC and PanD.

In one embodiment, the invention comprises a recombinant bacteria thatdoes not require supplementation with any vitamins.

In a second aspect, the invention provides a nucleic acid encoding oneor more enzymes in one or more biosynthesis pathway which produces oneor more vitamins.

In one embodiment, the nucleic acid encodes two or more enzymes. In oneembodiment, the nucleic acids of the invention encode 3, 4, 5 or 6 suchenzymes.

In one embodiment, the one or more vitamin is chosen from the groupcomprising thiamine, pathothenate, riboflavin, nicotinic acid,pyridoxine, biotin, folic acid, and cyanocobalamine. In one particularembodiment, the vitamin is thiamine (B1) or panthothenate (B5).

In one embodiment, the one or more of the enzymes is as herein afterdescribed.

In third aspect, the invention provides a nucleic acid construct orvector comprising one or more nucleic acid of the second aspect.

In one particular embodiment, the nucleic acid construct or vector is anexpression construct or vector. In one particular embodiment, theexpression construct or vector is a plasmid.

In a fourth aspect, the invention provides host organisms comprising anyone or more of the nucleic acids of the second aspect or vectors orconstructs of the third aspect.

In a fifth aspect, the invention provides a composition comprising anexpression construct or vector as referred to in the third aspect of theinvention and a methylation construct or vector.

Preferably, the composition is able to produce a recombinantmicroorganism according to the first aspect of the invention.

In one particular embodiment, the expression construct/vector and/or themethylation construct/vector is a plasmid.

In a sixth aspect, the invention provides a method of producing one ormore products by microbial fermentation comprising fermenting asubstrate using a recombinant microorganism of the first aspect of theinvention.

In one embodiment, the substrate is chosen from a substrate comprisingCO, carbon dioxide and hydrogen, glycerol, fatty acids, starch,molasses, pentoses and hexoses sugars, biomass.

In one particular embodiment, the substrate is a substrate comprisingCO. In this embodiment, the methods of the invention may be used toreduce the total atmospheric carbon emissions from an industrialprocess.

Preferably, the fermentation comprises the steps of fermenting asubstrate in a bioreactor to produce the one or more products using arecombinant microorganism of the invention.

In one embodiment the method comprises the steps of:

-   -   (a) providing a substrate to a bioreactor containing a culture        of one or more microorganism of the invention; and    -   (b) anaerobic fermentation by the culture in the bioreactor to        produce the one or more products.

In one embodiment the method comprises the steps of:

-   -   (a) capturing CO-containing gas produced as a result of the        industrial process, before the gas is released into the        atmosphere;    -   (b) the anaerobic fermentation of the CO-containing gas to        produce the one or more products by a culture containing one or        more microorganism of the first aspect of the invention.

In particular embodiments of the method aspects, the microorganism ismaintained in an aqueous culture medium.

In particular embodiments of the method aspects, the fermentation of thesubstrate takes place in a bioreactor.

In a particular embodiment, the substrate comprising CO is a gaseoussubstrate comprising CO. In one embodiment, the substrate comprises anindustrial waste gas. In certain embodiments, the gas is steel millwaste gas or syngas.

In one embodiment, the substrate will typically contain a majorproportion of CO, such as at least about 20% to about 100% CO by volume,from 20% to 70% CO by volume, from 30% to 60% CO by volume, and from 40%to 55% CO by volume. In particular embodiments, the substrate comprisesabout 25%, or about 30%, or about 35%, or about 40%, or about 45%, orabout 50% CO, or about 55% CO, or about 60% CO by volume.

In certain embodiments the methods further comprise the step ofrecovering one or more of the one or more products from the fermentationbroth.

In certain embodiments, the one or more products include ethanol,acetate, butanol, isopropanol, one or more vitamins, acetone,2,3-butanediol.

In one particular embodiment, the one or more product is one or morevitamin.

In one embodiment, the one or more product (in one embodiment, avitamin) is recovered from the fermentation and passed to one or morefurther bioreactor to support the growth of, or support the fermentationof a substrate by, one or more second microorganism.

In one embodiment, the one or more second microorganism is amicroorganism which is unable to produce, or unable to produce atsufficient levels, the one or more product (in one embodiment, avitamin) and requires its growth or fermentation media to besupplemented with the one or more product (in one embodiment, a vitamin)to ensure or maintain growth or fermentation, or to increase theefficiency of growth or fermentation.

In one embodiment, the methods of the invention may further comprisefermentation of a substrate by the one or more second microorganism toproduce one or more products. In one embodiment, the one or moreproducts may then be recovered from the fermentation broth.

In a seventh aspect, the invention provides one or more productsproduced by the method of the sixth aspect. In certain embodiments, theone or more products include ethanol, acetate, butanol, isopropanol, oneor more vitamins, acetone, 2,3-butanediol.

In an eighth aspect, the invention provides a method for the productionof a microorganism of the first aspect of the invention comprisingtransforming a parental microorganism with one or more exogenous nucleicacid which is adapted to express one or more enzyme in one or morevitamin biosynthesis pathway which produces one or more vitamins.

In certain embodiments, the one or more vitamins and enzymes are asherein after described.

In a nineth aspect, the invention provides a method for the selection ofmicroorganism A in a mixed population of microorganisms, whereinmicroorganism A is a recombinant microorganism comprising at least oneexogenous nucleic acid which is adapted to express one or more enzymesin one or more vitamin biosynthesis pathway which produces one or morevitamin(s) which are needed for the growth of the microorganism, suchthat the microorganism A can produce the one or more vitamin(s), themethod comprising subjecting the mixed population of microorganisms togrowth conditions including a media which lacks the one or morevitamin(s).

In one embodiment, the one or more vitamin is essential for growth ofthe microorganism.

In one embodiment, the at least one exogenous nucleic acid encodes oneor more gene encoding one or more enzymes in one or more vitaminbiosynthesis pathway.

In one embodiment, the one or more vitamin is as herein beforedescribed.

In one embodiment, the one or more of the enzyme is as herein afterdescribed.

In one embodiment, the media is chosen from any appropriate mediasuitable for culturing one or more microorganism.

In one embodiment, the method is performed to distinguish betweenrecombinant and non-recombinant microorganisms during the process ofproducing recombinant microorganisms.

In another embodiment, the method is performed to select againstcontaminating microorganisms during growth of, and/or fermentation of asubstrate by, microorganism A.

In a tenth aspect, the invention provides a means of preventing thegrowth of one or more undesirable microorganism in a microbial cultureor a fermentation broth, wherein the microbial culture or fermentationbroth comprises microorganism A and a nutrient media, whereinmicroorganism A is a recombinant microorganism comprising at least oneexogenous nucleic acid which is adapted to express one or more enzymesin one or more vitamin biosynthesis pathway which produces one or morevitamin(s) which is needed for the growth of microorganism A and theundesirable microorganism(s), such that the microorganism A can producethe one or more vitamin(s), wherein the media lacks the one or morevitamin(s) which is needed for the growth of the microorganisms.

In an eleventh aspect, the invention provides a method for the selectivegrowth or culture of a microorganism A, and wherein microorganism A is arecombinant microorganism comprising at least one exogenous nucleic acidwhich is adapted to express one or more enzymes in one or more vitaminbiosynthesis pathway which produces one or more vitamin(s) which areneeded for the growth of the microorganism, such that the microorganismA can produce the one or more vitamin(s), and wherein the growth orculture media lacks the one or more vitamin(s).

In one embodiment, the conditions select against the growth of one ormore undesirable microorganism(s).

In a twelfth aspect, the invention provides a method for the productionof one or more products by microbial fermentation of a substrate by amicroorganism A, wherein microorganism A is a recombinant microorganismcomprising at least one exogenous nucleic acid which is adapted toexpress one or more enzymes in one or more vitamin biosynthesis pathwaywhich produces one or more vitamin(s) which are needed for the growth ofthe microorganism, such that the microorganism A can produce the one ormore vitamin(s), and wherein fermentation occurs in or on a growth mediawhich lacks the one or more vitamin(s).

In one embodiment, the conditions select for growth of microorganism Aand against the growth of one or more undesirable microorganism(s).

The microorganisms of the nineth, tenth, eleventh and twelfth aspects ofthe invention may be chosen from any microorganism of interest, and arenot limited to anaerobes. However, in one embodiment they are chosenfrom the group of anaerobic microorganisms. In one embodiment, they arechosen from the group of carboxydotrophic acetogens.

Microbes and Growing Them

Isolated, genetically engineered, carboxydotrophic, acetogenic bacteriaare contemplated. These may be prototrophic for thiamine, pantothenate,riboflavin, nicotinic acid, pyridoxine, biotin, folic acid, and/orcyanocobalamine by virtue of an exogenous biosynthetic gene in thebiosynthetic pathway for the vitamin. For example an exogenous thiC geneand/or an exogenous panBCD gene cluster may be used to convert anauxotroph to a prototroph. In various embodiments the bacteria may beClostridium autoethanogenum, Clostridium ljungdahlii, Clostridiumragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridiumscatologenes, Butyribacterium methylotrophicum, Acetobacterium woodii,Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorellathermoacetica, Moorella thermautotrophica, Oxobacter pfennigii, orThermoanaerobacter kiuvi. In certain embodiments they are Clostridiumbacteria such as C. ljundahlii, C. autoethanogenum, C. ragsdalei, and C.carboxidivorans. Optionally the bacterium is unable to convert1-(5′-Phosphoribosyl)-5-aminoimidazole ribonucleotide (AIR) to4-amino-5-hydroxymethyl-2-methylpyrimidine in the absence of saidexogenous thiC gene. An exogenous thiC gene from C. ragsdalei iscontemplated and exemplified below. The exogenous thiC gene may be on aplasmid, such that the bacterium is auxotrophic for thiamine when curedof a plasmid. Alternatively it is contemplated that the bacterium isauxotrophic for pantothenate when cured of a plasmid. In certainembodiments, isolated, generically engineered, carboxydotrophic bacteriaare contemplated. In certain embodiments the bacteria maybe C.beijerinckii, C. acetobutylicum, C. saccharoperbutylacetonicum, C.phytofermentans, C. thermocellum, C. cellulovorans, C. cellulolyticum.

One particular exogenous panBCD gene cluster which may be used is fromC. beijerenckei.

The isolated genetically engineered carboxydotrophic acetogenic bacteriamay be cultured by growing them in a medium comprising a gaseous carbonsource; the carbon source may comprise CO. Similarly, the bacteria maybe cultured in a medium comprising an energy source which comprises CO.It is contemplated that the culture may be strictly anaerobic. It isfurther contemplated that if the bacterium comprises an exogenous thiCgene that the the medium can be devoid of thiamine. In some embodimentsthe bacterium will comprise an exogenous panBCD gene cluster and themedium will be devoid of pantothenate. In other embodiments thebacterium will comprises one or more exogenous genes in a biosyntheticpathway and the medium will be devoid of the product of thecorresponding biosynthetic pathway. In some embodiments, the carbonsource may comprise an industrial waste stream, such as waste gas fromferrous metal products manufacturing such as steel mill waste gas, wastegas from non-ferrous products manufacturing, waste gas from petroleumrefining processes, waste gas from gasification of coal, waste gas fromelectric power production, waste gas from carbon black production, wastegas from ammonia production, waste gas from methanol production, wastegas from coke manufacturing, and syngas. Automobile exhaust fumes mayalso be used as a carbon source.

CO Conversion Processes

One embodiment contemplated is a process for converting CO in aCO-containing substrate into higher molecular weight products. Theprocess comprises passing the CO-containing substrate to a bioreactorcontaining a culture of carboxydotrophic acetogenic bacteria in aculture medium such that the bacteria convert the CO to higher moleculeweight products; and recovering the higher molecular weight productsfrom the bioreactor. The carboxydotrophic acetogenic bacteria aregenetically engineered to express an enzyme in a biosynthetic pathway ofa nutrient that is absent from the culture medium. Optionally addednutrients may be provided to the culture medium for survival and/orgrowth of the carboxydotrophic acetogenic bacteria. When thecarboxydotrophic acetogenic bacteria are genetically engineered toexpress an enzyme in a biosynthetic pathway of a nutrient, that nutrientis absent from the added nutrients. Optionally the carboxydotrophicacetogenic bacteria are genetically engineered from parental bacteriathat are auxotrophic for the nutrient. In one alternative, the highermolecular weight products are selected from the group consisting ofalcohols, acids, diols, esters, ketones, and mixtures thereof. Inanother alternative, the higher molecular weight products are selectedfrom the group consisting of ethanol, acetone, 1-propanol, 2-propanol,1-butanol, 2-butanol, 1,4-butanediol, 2,3-butaendiol Methyl Ethyl Ketone(MEK), 3-hyrdoxypropionic acid, fatty acid. Terpenoids, 1,3-butadiene,3-hydroxybutyrate, 2-hydroxyisobutyric acid, acetic acid, and mixturesthereof. Optionally, a selective agent is added to the culture medium,such as an antibiotic to which the desired bacterium is resistant. Theantibiotic can be used to inhibit growth of undesired, contaminatingmicroorganisms, or bacteria that have lost the desired biosyntheticenzyme or pathway of enzymes. Notably antibiotic may not be necessaryand in some embodiments no exogenous antibiotic is in the culturemedium. The biosynthetic enzyme conferring prototrophy may exertsufficient selective pressure to maintain a culture of the desiredmicoorganisms. This may be a benefit in terms of cost savings,environmental protection, and human health in particular. Typically theculture medium is an aqueous mixture containing dissolved or undissolvedgasses. Typically the culturing conditions are maintained between atemperature from about 30° C. and about 37° C. and a pH from about 4 toless than 7. In one embodiment the CO-containing substrate ispre-treated to remove gaseous components other than CO. In someembodiments the nutrient may be produced in excess of the amountrequired by the bacteria in the culture. In such embodiments that excessnutrient may be collected as a product of the fermentation.

In one embodiment CO in a gaseous CO-containing substrate is converedinto higher molecular weight products. The gaseous CO-containingsubstrate is passed to a bioreactor containing a culture ofcarboxydotrophic acetogenic bacteria in a culture medium such that thebacteria convert the CO to higher molecule weight products. The highermolecular weight products are recovered from the bioreactor. Thecarboxydotrophic acetogenic bacteria are genetically engineered toexpress an enzyme in a biosynthetic pathway of a nutrient that is absentfrom the culture medium. Moreover, the carboxydotrophic acetogenicbacteria are prototrophic for thiamine and/or pantothenate by virtue ofan exogenous thiC gene and/or an exogenous panBCD gene cluster. Thenutrient is selected from the group consisting of thiamine andpantothenate.

Compositions as Used in the CO Conversion Processes

Another embodiment is a composition for converting CO in a CO-containingsubstrate into higher molecular weight products. The composition maycomprise carboxydotrophic acetogenic bacteria contained in an aqueousculture medium having a pH from about 4 to less than 7, and one or morenutrients for survival or growth of the carboxydotrophic acetogenicbacteria. The carboxydotrophic acetogenic bacteria are geneticallyengineered to express an enzyme in a biosynthetic pathway of a nutrientthat is absent from the culture media, such as thiamine or pantothenate.The composition may be in a container such as a bioreactor and typicallywill contain a gaseous carbon source comprising CO.

Other Methods

Another embodiment contemplated is a method for providing amicroorganism for use in reducing greenhouse gas emissions from anindustrial process. Genomic sequences of the microorganism are analyzedto determine whether a gene encoding an enzyme necessary in abiosynthetic pathway of an essential nutrient is lacking or defective.If a missing or defective enzyme is found, an exogenous (orheterologous) version of the gene is supplied to the microorganism bymeans of a gene transfer technique so that the microorganism becomesprototrophic for the essential nutrient. The heterologous gene may befrom a different species, a different genus, a different phylum, or evena different kingdom. Complementation of the genetic defect or lack willmake the microorganism prototrophic.

Yet another embodiment contemplated is a method for transferring anexogenous nucleic acid into a population of carboxydotrophic acetogenicbacteria which are auxotrophic for thiamine and/or pantothenate. Thebacteria are transformed with a first nucleic acid which comprises anexogenous thiC gene or an exogenous panBCD gene cluster operably linkedto a promoter.

Thiamine prototrophy and/or pantothenate prototrophy is selected foramong the transformed bacterial population. Optionally the bacteria maybe co-transformed with a second nucleic acid which comprises anexogenous or endogenous gene conferring a desired property whenexpressed in the bacterium. The desired property need not be selectable.The first and second nucleic acids may be on separate molecules or inthe same molecule. Optionally an additional step may be employed toscreen prototrophic, transformed bacteria for the presence of the firstnucleic acid. Under certain circumstances, it may be necessary and/ordesirable to treat the first and second nucleic acids to form methylatedfirst and second nucleic acids prior to the step of co-transforming.

Yet another embodiment uses prototrophy as a selectable marker on itsown for transformants, in the absence of any other selective agent suchas an antibiotic. The prototrophy may be for a vitamin, as shown below,or any other essential nutrient. The clean selection in the absence ofan antibiotic was unexpected. This type of selection can be used in anyany Clostridium described herein, as well as in other gram negative andgram positive bacteria, whether under aerobic or anaerobic growthconditions.

The invention may also be said broadly to consist in the parts, elementsand features referred to or indicated in the specification of theapplication, individually or collectively, in any or all combinations oftwo or more of said parts, elements or features, and where specificintegers are mentioned herein which have known equivalents in the art towhich the invention relates, such known equivalents are deemed to beincorporated herein as if individually set forth.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the present invention, which should beconsidered in all its novel aspects, will become apparent from thefollowing description, which is given by way of example only, withreference to the accompanying figures, in which:

FIG. 1: Growth and metabolite profile of LZ1561 in a continuous culturebetween days twenty and twenty eight.

FIG. 2: Genomic arrangement of Nucleotide sequence of C. ragsdaleithic/purF region, amplified by PCR.

FIG. 3: Plasmid map of pMTL85246-thiC-purF.

FIG. 4: Genomic arrangement of Nucleotide sequence of C. beijerickiipanBCD operon.

FIG. 5: Comparison of growth from C. autoethanogenum DSM23693 wild-typeand strain carrying plasmid pMTL85246-thiC-purF in absence of thiamine.

A sequence listing is part of this application.

DETAILED DESCRIPTION OF THE INVENTION

The following is a description of the present invention, includingpreferred embodiments thereof, given in general terms. The invention isfurther elucidated from the disclosure given under the heading“Examples” herein below, which provides experimental data supporting theinvention, specific examples of various aspects of the invention, andmeans of performing the invention.

The inventors have surprisingly identified that one or more gene(s) in abiosynthesis pathway for a vitamin which is needed for the survival of amicroorganism can be used as an effective selective marker to screen forcells transformed with exogenous nucleic acid(s) where the microorganismdoes not naturally contain or express the one or more gene(s).

This has a number of advantages, including obviating the need for theuse of standard selective markers and agents, such as antibiotics, whichare expensive, have limitations, and can be toxic to some desirablecells. It also has the benefit of further reducing the cost of thegrowth and fermentation of recombinant microorganisms as vitamins thatwould typically need to be added to growth and fermentation media can beomitted. Further, vitamins are themselves a valuable product, so inaddition to acting as a selection marker, the vitamins produced may berecovered and sold or used for other purposes.

As referred to herein, a “fermentation broth” is a culture mediumcomprising at least a nutrient media and bacterial cells.

As referred to herein, a “shuttle microorganism” is a microorganism inwhich a methyltransferase enzyme is expressed and is distinct from thedestination microorganism.

As referred to herein, a “destination microorganism” is a microorganismin which the genes included on an expression construct/vector areexpressed and is distinct from the shuttle microorganism.

The term “main fermentation product” is intended to mean the onefermentation product which is produced in the highest concentrationand/or yield.

The terms “increasing the efficiency”, “increased efficiency” and thelike, when used in relation to a fermentation process, include, but arenot limited to, increasing one or more of the rate of growth ofmicroorganisms catalysing the fermentation, the growth and/or productproduction rate at elevated product concentrations, the volume ofdesired product produced per volume of substrate consumed, the rate ofproduction or level of production of the desired product, and therelative proportion of the desired product produced compared with otherby-products of the fermentation.

The phrase “substrate comprising carbon monoxide” and like terms shouldbe understood to include any substrate in which carbon monoxide isavailable to one or more strains of bacteria for growth and/orfermentation, for example.

The phrase “gaseous substrate comprising carbon monoxide” and likephrases and terms includes any gas which contains a level of carbonmonoxide. In certain embodiments the substrate contains at least about20% to about 100% CO by volume, from 20% to 70% CO by volume, from 30%to 60% CO by volume, and from 40% to 55% CO by volume. In particularembodiments, the substrate comprises about 25%, or about 30%, or about35%, or about 40%, or about 45%, or about 50% CO, or about 55% CO, orabout 60% CO by volume.

While it is not necessary for the substrate to contain any hydrogen, thepresence of H₂ should not be detrimental to product formation inaccordance with methods of the invention. In particular embodiments, thepresence of hydrogen results in an improved overall efficiency ofalcohol production. For example, in particular embodiments, thesubstrate may comprise an approx 2:1, or 1:1, or 1:2 ratio of H₂:CO. Inone embodiment the substrate comprises about 30% or less H₂ by volume,20% or less H₂ by volume, about 15% or less H₂ by volume or about 10% orless H₂ by volume. In other embodiments, the substrate stream compriseslow concentrations of H₂, for example, less than 5%, or less than 4%, orless than 3%, or less than 2%, or less than 1%, or is substantiallyhydrogen free. The substrate may also contain some CO₂ for example, suchas about 1% to about 80% CO₂ by volume, or 1% to about 30% CO₂ byvolume. In one embodiment the substrate comprises less than or equal toabout 20% CO₂ by volume. In particular embodiments the substratecomprises less than or equal to about 15% CO₂ by volume, less than orequal to about 10% CO₂ by volume, less than or equal to about 5% CO₂ byvolume or substantially no CO₂.

In the description which follows, embodiments of the invention aredescribed in terms of delivering and fermenting a “gaseous substratecontaining CO”. However, it should be appreciated that the gaseoussubstrate may be provided in alternative forms. For example, the gaseoussubstrate containing CO may be provided dissolved in a liquid.Essentially, a liquid is saturated with a carbon monoxide containing gasand then that liquid is added to the bioreactor. This may be achievedusing standard methodology. By way of example, a microbubble dispersiongenerator (Hensirisak et. al. Scale-up of microbubble dispersiongenerator for aerobic fermentation; Applied Biochemistry andBiotechnology Volume 101, Number 3/October, 2002) could be used. By wayof further example, the gaseous substrate containing CO may be adsorbedonto a solid support. Such alternative methods are encompassed by use ofthe term “substrate containing CO” and the like.

In particular embodiments of the invention, the CO-containing gaseoussubstrate is an industrial off or waste gas. “Industrial waste or offgases” should be taken broadly to include any gases comprising COproduced by an industrial process and include gases produced as a resultof ferrous metal products manufacturing, non-ferrous productsmanufacturing, petroleum refining processes, gasification of coal,gasification of biomass, electric power production, carbon blackproduction, and coke manufacturing. Further examples may be providedelsewhere herein.

Unless the context requires otherwise, the phrases “fermenting”,“fermentation process” or “fermentation reaction” and the like, as usedherein, are intended to encompass both the growth phase and productbiosynthesis phase of the process. As will be described further herein,in some embodiments the bioreactor may comprise a first growth reactorand a second fermentation reactor. As such, the addition of metals orcompositions to a fermentation reaction should be understood to includeaddition to either or both of these reactors.

The term “bioreactor” includes a fermentation device consisting of oneor more vessels and/or towers or piping arrangement, which includes theContinuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR),Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, StaticMixer, or other vessel or other device suitable for gas-liquid contact.In some embodiments the bioreactor may comprise a first growth reactorand a second fermentation reactor. As such, when referring to theaddition of substrate to the bioreactor or fermentation reaction itshould be understood to include addition to either or both of thesereactors where appropriate.

“Exogenous nucleic acids” are nucleic acids which originate outside ofthe microorganism to which they are introduced. Exogenous nucleic acidsmay be derived from any appropriate source, including, but not limitedto, strains or species of microorganisms which differ from the organismto which they are to be introduced, or they may be artificially orrecombinantly created. The exogenous nucleic acids represent nucleicacid sequences not naturally present within the microorganism to whichthey are to be introduced and allow for the expression of a product notnaturally present within the microorganism. The exogenous nucleic acidmay be adapted to integrate into the genome of the microorganism towhich it is to be introduced or to remain in an extra-chromosomal state.Typically the exogenous nucleic acid is heterologous, coming from adifferent species, a different genus, or a different phylum or kingdomthan the recipient.

It should be appreciated that the invention may be practised usingnucleic acids whose sequence varies from the sequences specificallyexemplified herein provided they perform substantially the samefunction. For nucleic acid sequences that encode a protein or peptidethis means that the encoded protein or peptide has substantially thesame function. For nucleic acid sequences that represent promotersequences, the variant sequence will have the ability to promoteexpression of one or more genes. Such nucleic acids may be referred toherein as “functionally equivalent variants”. By way of example,functionally equivalent variants of a nucleic acid include allelicvariants, fragments of a gene, genes which include mutations (deletion,insertion, nucleotide substitutions and the like) and/or polymorphismsand the like. Homologous genes from other microorganisms may also beconsidered as examples of functionally equivalent variants of thesequences specifically exemplified herein. These include homologousgenes in species such as E. coli, Bacillus subtilis, Clostridiumbeijerinckii, details of which are publicly available on websites suchas Genbank or NCBI. The phrase “functionally equivalent variants” shouldalso be taken to include nucleic acids whose sequence varies as a resultof codon optimisation for a particular organism. “Functionallyequivalent variants” of a nucleic acid herein will preferably have atleast approximately 70%, preferably approximately 80%, more preferablyapproximately 85%, preferably approximately 90%, preferablyapproximately 95% or greater nucleic acid sequence identity with thenucleic acid identified.

It should also be appreciated that the invention may be practised usingpolypeptides whose sequence varies from the amino acid sequencesspecifically exemplified herein. These variants may be referred toherein as “functionally equivalent variants”. A functionally equivalentvariant of a protein or a peptide includes those proteins or peptidesthat share at least 40%, preferably 50%, preferably 60%, preferably 70%,preferably 75%, preferably 80%, preferably 85%, preferably 90%,preferably 95% or greater amino acid identity with the protein orpeptide identified and has substantially the same function as thepeptide or protein of interest. Such variants include within their scopefragments of a protein or peptide wherein the fragment comprises atruncated form of the polypeptide wherein deletions may be from 1 to 5,to 10, to 15, to 20, to 25 amino acids, and may extend from residue 1through 25 at either terminus of the polypeptide, and wherein deletionsmay be of any length within the region; or may be at an internallocation. Functionally equivalent variants of the specific polypeptidesherein should also be taken to include polypeptides expressed byhomologous genes in other species of bacteria, for example asexemplified in the previous paragraph.

“Substantially the same function” as used herein is intended to meanthat the nucleic acid or polypeptide is able to perform the function ofthe nucleic acid or polypeptide of which it is a variant. For example, avariant of an enzyme of the invention will be able to catalyse the samereaction as that enzyme. However, it should not be taken to mean thatthe variant has the same level of activity as the polypeptide or nucleicacid of which it is a variant.

One may assess whether a functionally equivalent variant hassubstantially the same function as the nucleic acid or polypeptide ofwhich it is a variant using any number of known methods. However, by wayof example, the methods outlined in Zhang et al (1997, J. Bacteriol.,179:3030-5), Lawhorn et al. (2004, Organic & Biomolecular Chemistry, 2:2538-46) may be used to assess functionality in respect of ThiC, Powers& Snell (1976, Biol. Chem. 251, 3786-3793) may be used to assessfunctionality in respect of PanB, Cronan et al. (1982, J. Bacteriol.149: 916-922) may be used to assess functionality in respect of PanC, orWilliamson (1985, Methods Enzymol. 113: 589-595) may be used to assessfunctionality in respect of PanD, or a genetic screen as outlined byLawhorn et al. (2004, J. Soc. Biol. Chem., 279: 43555-9) in respect ofthiamine genes may be used.

A “parental microorganism” is a microorganism used to generate arecombinant microorganism of the invention. The parental microorganismmay be one that occurs in nature (ie a wild type microorganism) or onethat has been previously modified but which does not express one or moreof the enzymes in the one or more vitamin biosynthesis pathway which isneeded for the growth of the microorganism. Accordingly, the recombinantmicroorganisms of the invention have been modified to express the one ormore enzymes that were not expressed in the parental microorganism.

The terms nucleic acid “constructs” or “vectors” and like terms shouldbe taken broadly to include any nucleic acid (including DNA and RNA)suitable for use as a vehicle to transfer genetic material into a cell.The terms should be taken to include plasmids, viruses (includingbacteriophage), cosmids and artificial chromosomes. Constructs orvectors may include one or more regulatory elements, an origin ofreplication, a multicloning site and/or a selectable marker. In oneparticular embodiment, the constructs or vectors are adapted to allowexpression of one or more genes encoded by the construct or vector.Nucleic acid constructs or vectors include naked nucleic acids as wellas nucleic acids formulated with one or more agents to facilitatedelivery to a cell (for example, liposome-conjugated nucleic acid, anorganism in which the nucleic acid is contained).

The invention is applicable to the production of “one or more products”by microbial fermentation. Reference to “one or more products” should betaken broadly to include any product which may be produced by microbialfermentation. However, by way of example, it includes, ethanol, acetate,butanol, isopropanol, one or more vitamins, acetone, 2,3-butanediol.

“Contaminating microorganisms” or “undesirable microorganisms” should betaken broadly to mean any microorganism that is not desired, for whatever reason.

When used in the context of the methods of the invention “preventinggrowth” and like terms should be taken broadly to include any level ofprevention or reduction in the level of growth of one or moremicroorganism. It should not be construed to mean that growth of amicroorganism is completed prevented, inhibited or stopped. However, ina preferred embodiment the growth of the microorganism is substantiallyprevented.

“Needed for growth” is to be taken to mean that the vitamin is requiredfor a desired level of growth, such that the absence of the vitamin in amedia in or on which a microorganism will grow is sufficient to selectfor a recombinant microorganism (able to make the vitamin) or selectagainst an undesirable or contaminating microorganism. In oneembodiment, a vitamin is “essential for growth” of the microorganism. Inthis case, without the vitamin, a microorganism will not substantiallygrow, or may die.

“Anaerobe” or “anaerobic microorganism” or like terms should be takenbroadly and to include both obligate and facultative anaerobes.

Recombinant Microorganisms

As discussed herein before, the invention provides a recombinantmicroorganism. The recombinant microorganism comprises at least oneexogenous nucleic acid which encodes one or more gene(s) in one or morevitamin biosynthesis pathway which produces a vitamin(s) which is neededfor growth of the microorganism. The recombinant microorganism isaccordingly able to produce the one or more vitamin(s). The recombinantmicroorganism is an anaerobe.

The recombinant microorganism is produced from a parental microorganism.

The parental microorganism may be chosen from any microorganism that islacking the one or more genes in the one or more vitamin biosynthesispathway.

In one embodiment, the vitamin is chosen from thiamine, pathothenate,riboflavin, nicotinic acid, pyridoxine, biotin, folic acid, andcyanocobalamine. In one particular embodiment, the vitamin is thiamine(B1) or panthothenate (B5).

The one or more enzymes may be any one which is involved in a vitaminbiosynthesis pathway. However, by way of example, the one or moreenzymes may be chosen from those listed in Tables 1 to 8 below.

TABLE 1 Thiamine (B1) biosynthesis: cysteine desulfurase [EC: 2.8.1.7]glycine oxidase [EC: 1.4.3.19] hydroxyethylthiazole kinase [EC:2.7.1.50] hydroxymethylpyrimidine [EC: 2.7.4.7]nucleoside-triphosphatase [EC: 3.6.1.15] phosphomethylpyrimidine kinase[EC: 2.7.1.49] selenocysteine lyase [EC: 4.4.1.16] sulfur carrierprotein ThiS adenylyltransferase [EC: 2.7.7.73] thiaminase [EC:3.5.99.2] thiamine biosynthesis protein ThiC [EC 4.1.99.17] thiaminebiosynthesis protein ThiG thiamine biosynthesis protein ThiH thiaminebiosynthesis protein ThiI thiamine kinase [EC: 2.7.1.89] thiaminepyridinylase [EC: 2.5.1.2] thiamine pyrophosphokinase [EC: 2.7.6.2]thiamine-monophosphate kinase [EC: 2.7.4.16] thiamine-phosphatepyrophosphorylase [EC: 2.5.1.3] thiamine-triphosphatase [EC: 3.6.1.28]

TABLE 2 Riboflavin (B2) biosynthesis: GTP cyclohydrolase II [EC:3.5.4.25] 2,5-diamino-6-(ribosylamino)-4(3H)-pyrimidinone 5′-phosphatereductase [EC: 1.1.1.302]2-amino-5-formylamino-6-ribosylaminopyrimidin-4(3H)-one 5′-monophosphatedeformylase [EC: 3.5.1.102] 3,4-dihydroxy 2-butanone 4-phosphatesynthase [EC: 4.1.99.12] 4-phytase/acid phosphatase [EC:3.1.3.2/3.1.3.26] 5,6-dimethylbenzimidazole synthase [EC: 1.14.99.40]5-amino-6-(5-phosphoribosylamino)uracil reductase [EC: 1.1.1.193]6,7-dimethyl-8-ribityllumazine synthase [EC: 2.5.1.78] acid phosphatase(class A) [EC: 3.1.3.2] acid phosphatase (class B) [EC: 3.1.3.2] acidphosphatase [EC: 3.1.3.2] aquacobalamin reductase/NAD(P)H-flavinreductase [EC: 1.5.1.41/1.16.1.3] biliverdin reductase/flavin reductase[EC: 1.5.1.30/1.3.1.24] diaminohydroxyphosphoribosylaminopyrimidinedeaminase/5-amino-6-(5- phosphoribosylamino)uracil reductase [EC:1.1.1.193/3.5.4.26] diaminohydroxyphosphoribosylaminopyrimidinedeaminase [EC: 3.5.4.26] ectonucleotidepyrophosphatase/phosphodiesterase family member 1/3 [EC:3.6.1.9/3.1.4.1] FAD synthetase [EC: 2.7.7.2] FMN reductase [EC:1.5.1.38] GTP cyclohydrolase II [EC: 3.5.4.25] GTP cyclohydrolase IIa[EC: 3.5.4.29] low molecular weight phosphotyrosine protein phosphatase[EC: 3.1.3.48/3.1.3.2] lysophosphatidic acid phosphatase type 6 [EC:3.1.3.2] FMN adenylyltransferase [EC: 2.7.7.2] riboflavin kinase [EC:2.7.1.26/EC: 2.7.1.161] riboflavin synthase [EC: 2.5.1.9]tartrate-resistant acid phosphatase type 5 [EC: 3.1.3.2] tRNApseudouridine synthase 8/2,5-diamino-6-(5-phospho-D-ribitylamino)-pyrimidin-4(3H)-one deaminase [EC: 5.4.99.—] tyrosinase[EC: 1.14.18.1]

TABLE 3 Nicotinic acid (B3) biosynthesis: 5′-nucleotidase [EC: 3.1.3.5]6-hydroxy-3-succinoylpyridine hydroxylase [EC: 3.7.1.—]6-hydroxynicotinate 3-monooxygenase [EC: 1.14.13.114] aldehyde oxidase[EC: 1.2.3.1] aspartate dehydrogenase [EC: 1.4.1.21] bifunctional NMNadenylyltransferase/nudix hydrolase [EC: 3.6.1.—2.7.7.1] ectonucleotidepyrophosphatase/phosphodiesterase family member 1/3 [EC: 3.6.1.93.1.4.1] enamidase [EC: 3.5.2.18] L-aspartate oxidase [EC: 1.4.3.16]maleamate amidohydrolase [EC: 3.5.1.107] maleate isomerase [EC: 5.2.1.1]NAD(P) transhydrogenase [EC: 1.6.1.1] NAD(P) transhydrogenase subunitalpha [EC: 1.6.1.2] NAD(P) transhydrogenase subunit beta [EC: 1.6.1.2]NAD+ diphosphatase [EC: 3.6.1.22] NAD+ kinase [EC: 2.7.1.23] NAD+nucleosidase [EC: 3.2.2.5] NAD+ synthase (glutamine-hydrolysing) [EC:6.3.5.1] NAD+ synthase [EC: 6.3.1.5] N-formylmaleamate deformylase [EC:3.5.1.106] nicotinamidase [EC: 3.5.1.19] nicotinamide mononucleotideadenylyltransferase [EC: 2.7.7.18 2.7.7.1] nicotinamideN-methyltransferase [EC: 2.1.1.1] nicotinamide phosphoribosyltransferase[EC: 2.4.2.12] nicotinamide riboside kinase [EC: 2.7.1.22]nicotinamide-nucleotide adenylyltransferase [EC: 2.7.7.1] nicotinatephosphoribosyltransferase [EC: 2.4.2.11] nicotinate-nucleotideadenylyltransferase [EC: 2.7.7.18] nicotinate-nucleotidepyrophosphorylase (carboxylating) [EC: 2.4.2.19] purine nucleosidase[EC: 3.2.2.1] purine-nucleoside phosphorylase [EC: 2.4.2.1]pyrazinamidase [EC: 3.5.1.—] quinolinate synthase [EC: 2.5.1.72]UDP-sugar diphosphatase [EC: 3.6.1.45]

TABLE 4 Panthothenate (B5) biosynthesis: 2-dehydropantoate 2-reductase[EC: 1.1.1.169] 3-methyl-2-oxobutanoate hydroxymethyltransferase PanB[EC: 2.1.2.11] 4′-phosphopantetheinyl transferase [EC: 2.7.8.—]4-phosphopantoate---beta-alanine ligase [EC: 6.3.2.36] acetolactatesynthase I/II/III large subunit [EC: 2.2.1.6] acetolactate synthaseI/III small subunit [EC: 2.2.1.6] acetolactate synthase II small subunit[EC: 2.2.1.6] acyl carrier protein phosphodiesterase [EC: 3.1.4.14]aspartate 1-decarboxylase PanD [EC: 4.1.1.11] beta-ureidopropionase [EC:3.5.1.6] biotin-[acetyl-CoA-carboxylase] ligase [EC: 6.3.4.15]branched-chain amino acid aminotransferase [EC: 2.6.1.42] dephospho-CoAkinase [EC: 2.7.1.24] dihydropyrimidinase [EC: 3.5.2.2]dihydropyrimidine dehydrogenase (NADP+) [EC: 1.3.1.2] dihydroxy-aciddehydratase [EC: 4.2.1.9] ectonucleotidepyrophosphatase/phosphodiesterase family member 1/3 [EC: 3.6.1.93.1.4.1] holo-[acyl-carrier protein] synthase [EC: 2.7.8.7] ketol-acidreductoisomerase [EC: 1.1.1.86] pantetheine hydrolase [EC: 3.5.1.92]pantetheine-phosphate adenylyltransferase [EC: 2.7.7.3] pantoate kinase[EC: 2.7.1.169] pantoate ligase/cytidylate kinase [EC: 2.7.4.14/6.3.2.1]pantoate--beta-alanine ligase PanC [EC: 6.3.2.1] phosphopantetheineadenylyltransferase/dephospho-CoA kinase [EC: 2.7.1.24 2.7.7.3]phosphopantothenate-cysteine ligase [EC: 6.3.2.5]phosphopantothenate--cysteine ligase [EC: 6.3.2.5]phosphopantothenoylcysteine decarboxylase [EC: 4.1.1.36] type Ipantothenate kinase [EC: 2.7.1.33] type II pantothenate kinase [EC:2.7.1.33] type III pantothenate kinase [EC: 2.7.1.33]

TABLE 5 Pyridoxin (B6) biosynthesis: 4-hydroxythreonine-4-phosphatedehydrogenase [EC: 1.1.1.262] aldehyde oxidase [EC: 1.2.3.1] D-erythrose4-phosphate dehydrogenase [EC: 1.2.1.72] erythronate-4-phosphatedehydrogenase [EC: 1.1.1.290] glutamine amidotransferase [EC: 2.6.—.—]phosphoserine aminotransferase [EC: 2.6.1.52] pyridoxal phosphatase [EC:3.1.3.74] pyridoxal phosphate phosphatase [EC: 3.1.3.74] pyridoxamine5′-phosphate oxidase [EC: 1.4.3.5] pyridoxine 4-dehydrogenase [EC:1.1.1.65] pyridoxine 5-phosphate synthase [EC: 2.6.99.2] pyridoxinebiosynthesis protein [EC: 4.—.—.—] pyridoxine kinase [EC: 2.7.1.35]threonine synthase [EC: 4.2.3.1]

TABLE 6 Biotin (B7) biosynthesis: 6-carboxyhexanoate--CoA ligase [EC:6.2.1.14] 8-amino-7-oxononanoate synthase [EC: 2.3.1.47]adenosylmethionine-8-amino-7-oxononanoate aminotransferase [EC:2.6.1.62] biotin synthetase [EC: 2.8.1.6]biotin-[acetyl-CoA-carboxylase] ligase [EC: 6.3.4.15] biotinidase [EC:3.5.1.12] biotin--protein ligase [EC: 6.3.4.15 6.3.4.11 6.3.4.106.3.4.9] dethiobiotin synthetase [EC: 6.3.3.3] type III pantothenatekinase [EC: 2.7.1.33]

TABLE 7 Folate (B9)/p-Aminobenzoate (B10) biosynthesis:2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase [EC:2.7.6.3] 4-amino-4-deoxychorismate lyase [EC: 2.6.1.85/EC: 4.1.3.38]6-pyruvoyl tetrahydrobiopterin synthase [EC: 4.2.3.12]6-pyruvoyltetrahydropterin 2′-reductase [EC: 1.1.1.220] alkalinephosphatase [EC: 3.1.3.1] dihydrofolate reductase [EC: 1.5.1.3]dihydrofolate synthase/folylpolyglutamate synthase [EC:6.3.2.17/6.3.2.12] dihydromonapterin reductase [EC: 1.5.1.—]dihydroneopterin aldolase/2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase [EC: 2.7.6.3/4.1.2.25]dihydroneopterin aldolase [EC: 4.1.2.25] dihydropteridine reductase [EC:1.5.1.34] dihydropteroate synthase [EC: 2.5.1.15] folylpolyglutamatesynthase [EC: 6.3.2.17] gamma-glutamyl hydrolase [EC: 3.4.19.9] GTPcyclohydrolase I [EC: 3.5.4.16] molybdenum cofactor biosynthesis proteinmolybdopterin synthase catalytic subunit [EC: 2.—.—.—] molybdopterinsynthase sulfur carrier subunit para-aminobenzoate synthetase [EC:2.6.1.85] para-aminobenzoate synthetase component I [EC: 2.6.1.85]para-aminobenzoate synthetase component II [EC: 2.6.1.85] sepiapterinreductase [EC: 1.1.1.153] thymidylate synthase [EC: 2.1.1.45]

TABLE 8 Cobalamin (B12) biosynthesis 5-aminolevulinate synthase [EC:2.3.1.37] 5-aminolevulinate: pyruvate aminotransferase [EC 2.6.1.43]adenosylcobinamide kinase/adenosylcobinamide-phosphateguanylyltransferase [EC: 2.7.1.156/2.7.7.62] adenosylcobinamide-GDPribazoletransferase [EC: 2.7.8.26] adenosylcobinamide-phosphate synthase[EC: 6.3.1.10] adenosylcobyric acid synthase [EC: 6.3.5.10]alpha-ribazole phosphatase [EC: 3.1.3.73] cob(I)alaminadenosyltransferase [EC: 2.5.1.17] cob(II)yrinic acid a,c-diamidereductase [EC: 1.16.8.1] cobalt-precorrin 5A hydrolase [EC: 3.7.1.12]cobalt-precorrin-5B (C1)-methyltransferase [EC: 2.1.1.195]cobalt-precorrin-7 (C15)-methyltransferase [EC: 2.1.1.196]cobaltochelatase CobN [EC: 6.6.1.2] cobyrinic acid a,c-diamide synthase[EC: 6.3.5.9/6.3.5.11] ferritin [EC: 1.16.3.1] glutamate-1-semialdehyde2,1-aminomutase [EC: 5.4.3.8] glutamyl-tRNA reductase [EC: 1.2.1.70]glutamyl-tRNA synthetase [EC: 6.1.1.17] hydroxymethylbilane synthase[EC: 2.5.1.61] nicotinate-nucleotide--dimethylbenzimidazolephosphoribosyltransferase [EC: 2.4.2.21] oxygen-independentcoproporphyrinogen III oxidase [EC: 1.3.99.22] porphobilinogen synthase[EC: 4.2.1.24] precorrin-2 dehydrogenase/sirohydrochlorin ferrochelatase[EC: 1.3.1.76/4.99.1.4] precorrin-2/cobalt-factor-2C20-methyltransferase [EC: 2.1.1.130/2.1.1.151] precorrin-3B synthase[EC: 1.14.13.83] precorrin-3B C17-methyltransferase [EC: 2.1.1.131]precorrin-4 C11-methyltransferase [EC: 2.1.1.133] precorrin-6X reductase[EC: 1.3.1.54] precorrin-6Y C5,15-methyltransferase [EC: 2.1.1.132]precorrin-8W decarboxylase [EC: 1.—.—.—] precorrin-8X methylmutase [EC:5.4.1.2] sirohydrochlorin cobaltochelatase [EC: 4.99.1.3]threonine-phosphate decarboxylase [EC: 4.1.1.81] uroporphyrinogendecarboxylase [EC: 4.1.1.37] uroporphyrinogen IIImethyltransferase/synthase [EC: 2.1.1.107 4.2.1.75]

In one particular embodiment, the parental microorganism lacks one ormore of the genes encoding the enzymes thiamine biosynthesis proteinThiC (EC 4.1.99.17), 3-methyl-2-oxobutanoate hydroxymethyltransferasePanB (EC 2.1.2.11), pantoate-beta-alanine ligase PanC (EC 6.3.2.1), andaspartate 1-decarboxylase Pan D (EC 4.1.1.11). In one embodiment, theparental microorganism lacks the gene encoding enzyme ThiC. In anotherembodiment, the parental microorganism lacks one or more enzymesencoding one or more or all of PanB, PanC and PanD.

While the invention is exemplified herein in respect of certain vitaminbiosynthesis pathways, it will be appreciated that other biosynthesispathways may be used. With knowledge of the present invention, analysisof any sequenced organism can be done using databases like the KyotoEncyclopedia of Genes and Genomes KEGG (http://www.genome.jp/kegg/;Kanehisa et al., 2012, Nucleic Acids Res. 40, D109-D114; Kanehisa andGoto, 2000, Nucleic Acids Res. 28, 27-30) or BioCyc (http://biocyc.org/;Caspi et al., 2010, Nucleic Acids Res. 38: D473-479) to identify geneswhich are present or missing from a particular biosynthesis pathway.

The parental microorganism may be chosen from any of the group ofanaerobic microorganism, in one embodiment anaerobic bacteria.

In one embodiment, the microorganism is selected from the groupcomprising the genera Clostridium, Eubacterium, Peptostreptococcus,Peptococcus, Actinomyces, Lactobacillus, Bifidobacterium,Propionibacterium, Bacteriodes, Fusobacterium, Campylobacter, orVeillonella.

In one particular embodiment, the parental microorganism is selectedfrom the group of carboxydotrophic acetogenic bacteria. In certainembodiments the microorganism is selected from the group comprisingClostridium autoethanogenum, Clostridium ljungdahlii, Clostridiumragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridiumscatologenes, Clostridium aceticum, Clostridium formicoaceticum,Clostridium magnum, Butyribacterium methylotrophicum, Acetobacteriumwoodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum,Moorella thermoacetica, Moorella thermautotrophica, Sporomusa ovata,Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacter pfennigii, andThermoanaerobacter kiuvi.

In one particular embodiment, the parental microorganism is selectedfrom the cluster of ethanologenic, acetogenic Clostridia comprising thespecies C. autoethanogenum, C. ljungdahlii, and C. ragsdalei and relatedisolates. These include but are not limited to strains C.autoethanogenum JAI-1^(T) (DSM10061) [Abrini J, Naveau H, Nyns E-J:Clostridium autoethanogenum, sp. nov., an anaerobic bacterium thatproduces ethanol from carbon monoxide. Arch Microbiol 1994, 4: 345-351],C. autoethanogenum LBS1560 (DSM19630) [Simpson S D, Forster R L, Tran PT, Rowe M J, Warner I L: Novel bacteria and methods thereof.International patent 2009, WO/2009/064200], C. autoethanogenum LBS1561(DSM23693), C. ljungdahlii PETC^(T) (DSM13528=ATCC 55383) [Tanner R S,Miller L M, Yang D: Clostridium ljungdahlii sp. nov., an AcetogenicSpecies in Clostridial rRNA Homology Group I. Int J Syst Bacteriol 1993,43: 232-236], C. ljungdahlii ERI-2 (ATCC 55380) [Gaddy J L: Clostridiumstain which produces acetic acid from waste gases. US patent 1997, U.S.Pat. No. 5,593,886], C. ljungdahlii C-01 (ATCC 55988) [Gaddy J L,Clausen E C, Ko C-W: Microbial process for the preparation of aceticacid as well as solvent for its extraction from the fermentation broth.US patent, 2002, U.S. Pat. No. 6,368,819], C. ljungdahlii O-52 (ATCC55989) [Gaddy J L, Clausen E C, Ko C-W: Microbial process for thepreparation of acetic acid as well as solvent for its extraction fromthe fermentation broth. US patent, 2002, U.S. Pat. No. 6,368,819], C.ragsdalei P11^(T) (ATCC BAA-622) [Huhnke R L, Lewis R S, Tanner R S:Isolation and Characterization of novel Clostridial Species.International patent 2008, WO 2008/028055], related isolates such as “C.coskatii” [Zahn et al—Novel ethanologenic species Clostridium coskatii(US Patent Application number US20110229947)] and “Clostridium sp.”(Tyurin et al., 2012, J. Biotech Res. 4: 1-12), or mutated strains suchas C. ljungdahlii OTA-1 (Tirado-Acevedo O. Production of Bioethanol fromSynthesis Gas Using Clostridium ljungdahlii. PhD thesis, North CarolinaState University, 2010). These strains form a subcluster within theClostridial rRNA cluster I, and their 16S rRNA gene is more than 99%identical with a similar low GC content of around 30%. However, DNA-DNAreassociation and DNA fingerprinting experiments showed that thesestrains belong to distinct species [Huhnke R L, Lewis R S, Tanner R S:Isolation and Characterization of novel Clostridial Species.International patent 2008, WO 2008/028055].

All species of this cluster have a similar morphology and size(logarithmic growing cells are between 0.5-0.7×3-5 μm), are mesophilic(optimal growth temperature between 30-37° C.) and strictly anaerobe[Tanner R S, Miller L M, Yang D: Clostridium ljungdahlii sp. nov., anAcetogenic Species in Clostridial rRNA Homology Group I. Int J SystBacteriol 1993, 43: 232-236; Abrini J, Naveau H, Nyns E-J: Clostridiumautoethanogenum, sp. nov., an anaerobic bacterium that produces ethanolfrom carbon monoxide. Arch Microbiol 1994, 4: 345-351; Huhnke R L, LewisR S, Tanner R S: Isolation and Characterization of novel ClostridialSpecies. International patent 2008, WO 2008/028055]. Moreover, they allshare the same major phylogenetic traits, such as same pH range (pH4-7.5, with an optimal initial pH of 5.5-6), strong autotrophic growthon CO containing gases with similar growth rates, and a similarmetabolic profile with ethanol and acetic acid as main fermentation endproduct, and small amounts of 2,3-butanediol and lactic acid formedunder certain conditions. [Tanner R S, Miller L M, Yang D: Clostridiumljungdahlii sp. nov., an Acetogenic Species in Clostridial rRNA HomologyGroup I. Int J Syst Bacteriol 1993, 43: 232-236; Abrini J, Naveau H,Nyns E-J: Clostridium autoethanogenum, sp. nov., an anaerobic bacteriumthat produces ethanol from carbon monoxide. Arch Microbiol 1994, 4:345-351; Huhnke R L, Lewis R S, Tanner R S: Isolation andCharacterization of novel Clostridial Species. International patent2008, WO 2008/028055]. Indole production was observed with all threespecies as well. However, the species differentiate in substrateutilization of various sugars (e.g. rhamnose, arabinose), acids (e.g.gluconate, citrate), amino acids (e.g. arginine, histidine), or othersubstrates (e.g. betaine, butanol). Moreover some of the species werefound to be auxotroph to certain vitamins (e.g. thiamine, biotin) whileothers were not.

The strains of this cluster are defined by common characteristics,having both a similar genotype and phenotype, and they all share thesame mode of energy conservation and fermentative metabolism. Thestrains of this cluster lack cytochromes and conserve energy via an Rnfcomplex.

All strains of this cluster have a genome size of around 4.2 MBp (Köpkeet al., 2010) and a GC composition of around 32% mol (Abrini et al.,1994; Köpke et al., 2010; Tanner et al., 1993) (WO 2008/028055; USpatent 2011/0229947), and conserved essential key gene operons encodingfor enzymes of Wood-Ljungdahl pathway (Carbon monoxide dehydrogenase,Formyl-tetrahydrofolate synthetase, Methylene-tetrahydrofolatedehydrogenase, Formyl-tetrahydrofolate cyclohydrolase,Methylene-tetrahydrofolate reductase, and Carbon monoxidedehydrogenase/Acetyl-CoA synthase), hydrogenase, formate dehydrogenase,Rnf complex (rnfCDGEAB), pyruvate:ferredoxin oxidoreductase,aldehyde:ferredoxin oxidoreductase (Köpke et al., 2010, 2011). Theorganization and number of Wood-Ljungdahl pathway genes, responsible forgas uptake, has been found to be the same in all species, despitedifferences in nucleic and amino acid sequences (Köpke et al., 2011).

Reduction of carboxylic acids into their corresponding alcohols has beenshown in a range of these organisms (Perez, Richter, Loftus, & Angenent,2012).

The traits described are therefore not specific to one organism like C.autoethanogenum or C. ljungdahlii, but rather general traits forcarboxydotrophic, ethanol-synthesizing Clostridia. Thus, the inventioncan be anticipated to work across these strains, although there may bedifferences in performance.

The recombinant carboxydotrophic acetogenic microorganisms of theinvention may be prepared from a parental carboxydotrophic acetogenicmicroorganism and one or more exogenous nucleic acids using any numberof techniques known in the art for producing recombinant microorganisms.By way of example only, transformation (including transduction ortransfection) may be achieved by electroporation, electrofusion,ultrasonication, polyethylene glycol-mediated transformation,conjugation, or chemical and natural competence. Suitable transformationtechniques are described for example in Sambrook J, Fritsch E F,Maniatis T: Molecular Cloning: A laboratory Manual, Cold Spring HarbourLabrotary Press, Cold Spring Harbour, 1989.

Electroporation has been described for several carboxydotrophicacetogens as C. ljungdahlii (Köpke et al., 2010; Leang, Ueki, Nevin, &Lovley, 2012) (PCT/NZ2011/000203; WO2012/053905), C. autoethanogenum(PCT/NZ2011/000203; WO2012/053905), Acetobacterium woodii (Strätz,Sauer, Kuhn, & Dürre, 1994) or Moorella thermoacetica (Kita et al.,2012) and is a standard method used in many Clostridia such as C.acetobutylicum (Mermelstein, Welker, Bennett, & Papoutsakis, 1992), C.cellulolyticum (Jennert, Tardif, Young, & Young, 2000) or C.thermocellum (M V Tyurin, Desai, & Lynd, 2004).

Electrofusion has been described for acetogenic Clostridium sp. MT351(Tyurin and Kiriukhin, 2012).

Prophage induction has been described for carboxydotrophic acetogen aswell in case of C. scatologenes (Prasanna Tamarapu Parthasarathy, 2010,Development of a Genetic Modification System in Clostridium scatologenesATCC 25775 for Generation of Mutants, Masters Project Western KentuckyUniversity).

Conjugation has been described as method of choice for acetogenClostridium difficile (Herbert, O'Keeffe, Purdy, Elmore, & Minton, 2003)and many other Clostridia including C. acetobuylicum (Williams, Young, &Young, 1990). In one embodiment, the parental strain uses CO as its solecarbon and energy source.

In one embodiment the parental microorganism is Clostridiumautoethanogenum or Clostridium ljungdahlii. In one particularembodiment, the microorganism is Clostridium autoethanogenum DSM23693.In another particular embodiment, the microorganism is Clostridiumljungdahlii DSM13528 (or ATCC55383).

In one particular embodiment, the parental microorganism is Clostridiumautoethanogenum, the biosynthesis pathway is for thiamine orpanthothenate and the parental microorganism lacks the genes thiC or oneor more of panB, panC and panD.

In one particular embodiment, the recombinant microorganism is adaptedto express one or more enzymes in a vitamin biosynthesis pathway whichproduces a vitamin(s) which is needed for the growth of themicroorganism and which are not naturally present in the parentalmicroorganism.

The microorganism may be adapted to express the one or more enzymes byany number of recombinant methods including, for example, introducing anexogenous nucleic acid encoding and adapted to express an enzyme notnaturally present within the parental microorganism.

The vitamins and enzymes of use in the recombinant microorganisms of theinvention are defined elsewhere herein.

In one embodiment, the microorganism comprises one or more exogenousnucleic acids encoding and adapted to express one or more of the enzymesreferred to elsewhere herein. In one embodiment, the microorganismscomprise one or more exogenous nucleic acid encoding and adapted toexpress at least two of the enzymes. In other embodiments, themicroorganism comprises one or more exogenous nucleic acid encoding andadapted to express 3, 4, 5, or 6 of the enzymes.

The microorganism may comprise one or more exogenous nucleic acids.Where it is desirable to transform the parental microorganism with twoor more genetic elements (such as) they may be contained on one or moreexogenous nucleic acids.

In one embodiment, the one or more exogenous nucleic acid is a nucleicacid construct or vector, in one particular embodiment a plasmid,encoding one or more of the enzymes referred to herein in anycombination.

The exogenous nucleic acids may remain extra-chromosomal upontransformation of the parental microorganism or preferably intergrateinto the genome of the parental microorganism. Accordingly, they mayinclude additional nucleotide sequences adapted to assist integration(for example, a region which allows for homologous recombination andtargeted integration into the host genome) or expression and replicationof an extrachromosomal construct (for example, origin of replication,promoter and other regulatory elements or sequences).

In one embodiment, the exogenous nucleic acids encoding one or enzymesas mentioned herein before will further comprise a promoter adapted topromote expression of the one or more enzymes encoded by the exogenousnucleic acids. In one embodiment, the promoter is a constitutivepromoter that is preferably highly active under appropriate fermentationconditions. Inducible promoters could also be used. In preferredembodiments, the promoter is selected from the group comprisingWood-Ljungdahl gene cluster and Phosphotransacetylase/Acetate kinasepromoters. It will be appreciated by those of skill in the art thatother promoters which can direct expression, preferably a high level ofexpression under appropriate fermentation conditions, would be effectiveas alternatives to the exemplified embodiments.

In one embodiment, the exogenous nucleic acid is an expression plasmid.

Nucleic Acids

The invention also provides nucleic acids and nucleic acid constructs ofuse in generating a recombinant microorganism of the invention.

The nucleic acids comprise sequences encoding one or more enzymes of oneor more vitamin biosynthesis pathway which when expressed in amicroorganism allows the microorganism to produce a vitamin which isneeded for the growth of the microorganism. In one particularembodiment, the invention provides a nucleic acid encoding two or moreenzymes. In one embodiment, the nucleic acids of the invention encode 3,4, 5 or 6 such enzymes.

A nucleic acid of the invention encodes one or more enzyme in a vitaminbiosynthesis pathway. In one particular embodiment, a nucleic acid ofthe invention encodes one or more enzyme in the biosynthesis pathway ofone or more of Thiamine, pathothenate, riboflavin, nicotinic acid,pyridoxine, biotin, folic acid, and cyanocobalamine. In one embodiment,a nucleic acid of the invention encodes one or more of the enzymeslisted in tables 3 to 10 herein before.

In one particular embodiment, a nucleic acid of the invention encodesone or more enzymes in the thiamine biosynthesis pathway. In oneembodiment, the nucleic acid encodes ThiC.

In another embodiment, a nucleic acid of the invention encodes one ormore enzymes in the panthothenate pathway. In one particular embodiment,a nucleic acid encodes one or more or all of panB, panC or panD.

Skilled persons will readily appreciate nucleic acids sequences encodingthe enzymes or functionally equivalent variants thereof which are of usein the invention, having regard to the information contained herein, inGenBank and other databases, and the genetic code. However, by way ofexample only, exemplary amino acid sequences and nucleic acid sequencesencoding enzymes of relevance to the invention may be obtained fromdatabases such as the NCBI, KEGG and BRENDA databases, for example.

By way of example only, in one embodiment, ThiC has the sequence of SEQID No. 3, or is a functionally equivalent variant thereof. By way offurther example, in one embodiment, panB has the sequence ofYP_(—)001309722.1 (GenBank) or is a functionally equivalent variantthereof, panC has the sequence of YP_(—)001309721.1 (GenBank) or is afunctionally equivalent variant thereof, and panD has the sequence ofYP_(—)001309720.1 (GenBank) or is a functionally equivalent variantthereof.

Again, by way of example only, in one embodiment, a nucleic acidencoding ThiC has the sequence of SEQ ID No. 2, or is a functionallyequivalent variant thereof. By way of further example, in oneembodiment, a nucleic acid encoding panB has the sequence ofCbei_(—)2610; Gene ID: 5293811 or is a functionally equivalent variantthereof, a nucleic acid encoding panC has the sequence of Cbei_(—)2609;Gene ID: 5293810 or is a functionally equivalent variant thereof, and anucleic acid encoding panD has the sequence of Cbei_(—)2608; Gene ID:5293809 or is a functionally equivalent variant thereof.

In one embodiment, the nucleic acids of the invention will furthercomprise a promoter. In one embodiment, the promoter allows forconstitutive expression of the genes under its control. However,inducible promoters may also be employed. Persons of skill in the artwill readily appreciate promoters of use in the invention. Preferably,the promoter can direct a high level of expression under appropriatefermentation conditions. In a particular embodiment a Wood-Ljungdahlcluster promoter is used. In another embodiment, aPhosphotransacetylase/Acetate kindase promoter is used. In anotherembodiment a pyruvate:ferredoxin oxidoreductase promoter, an Rnf complexoperon promoter or an ATP synthase operon promoter. In one particularembodiment, the promoter is from C. autoethanogenum.

The nucleic acids of the invention may remain extra-chromosomal upontransformation of a parental microorganism or may preferably be adaptedfor intergration into the genome of the microorganism. Accordingly,nucleic acids of the invention may include additional nucleotidesequences adapted to assist integration (for example, a region whichallows for homologous recombination and targeted integration into thehost genome) or stable expression and replication of an extrachromosomalconstruct (for example, origin of replication, promoter and otherregulatory sequences).

In one embodiment, the nucleic acid is nucleic acid construct or vector.In one particular embodiment, the nucleic acid construct or vector is anexpression construct or vector, however other constructs and vectors,such as those used for cloning are encompassed by the invention. In oneparticular embodiment, the expression construct or vector is a plasmid.

It will be appreciated that an expression construct/vector of thepresent invention may contain any number of regulatory elements inaddition to the promoter as well as additional genes suitable forexpression of further proteins if desired. In one embodiment theexpression construct/vector includes one promoter. In anotherembodiment, the expression construct/vector includes two or morepromoters. In one particular embodiment, the expression construct/vectorincludes one promoter for each gene to be expressed. In one embodiment,the expression construct/vector includes one or more ribosomal bindingsites, preferably a ribosomal binding site for each gene to beexpressed.

It will be appreciated by those of skill in the art that the nucleicacid sequences and construct/vector sequences described herein maycontain standard linker nucleotides such as those required for ribosomebinding sites and/or restriction sites. Such linker sequences should notbe interpreted as being required and do not provide a limitation on thesequences defined.

Nucleic acids and nucleic acid constructs, including expressionconstructs/vectors of the invention may be constructed using any numberof techniques standard in the art. For example, chemical synthesis orrecombinant techniques may be used. Such techniques are described, forexample, in Sambrook et al (Molecular Cloning: A laboratory manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Furtherexemplary techniques are described in the Examples section herein after.Essentially, the individual genes and regulatory elements will beoperably linked to one another such that the genes can be expressed toform the desired proteins. Suitable vectors for use in the inventionwill be appreciated by those of ordinary skill in the art. However, byway of example, the following vectors may be suitable: pMTL80000vectors, pIMP1, pJIR750, and the plasmids exemplified in the Examplessection herein after.

It should be appreciated that nucleic acids of the invention may be inany appropriate form, including RNA, DNA, or cDNA.

The invention also provides host organisms, particularly microorganisms,and including viruses, bacteria, and yeast, comprising any one or moreof the nucleic acids described herein.

The one or more exogenous nucleic acids may be delivered to a parentalmicroorganism as naked nucleic acids or may be formulated with one ormore agents to facilitate the transformation process (for example,liposome-conjugated nucleic acid, an organism in which the nucleic acidis contained). The one or more nucleic acids may be DNA, RNA, orcombinations thereof, as is appropriate. Restriction inhibitors may beused in certain embodiments; see, for example Murray, N. E. et al.(2000) Microbial. Molec. Biol. Rev. 64, 412.)

The microorganisms of the invention may be prepared from a parentalmicroorganism and one or more exogenous nucleic acids using any numberof techniques known in the art for producing recombinant microorganisms.By way of example only, transformation (including transduction ortransfection) may be achieved by electroporation, ultrasonication,polyethylene glycol-mediated transformation, chemical or naturalcompetence, or conjugation. Suitable transformation techniques aredescribed for example in, Sambrook J, Fritsch E F, Maniatis T: MolecularCloning: A laboratory Manual, Cold Spring Harbour Labrotary Press, ColdSpring Harbour, 1989.

In certain embodiments, due to the restriction systems which are activein the microorganism to be transformed, it is necessary to methylate thenucleic acid to be introduced into the microorganism. This can be doneusing a variety of techniques, including those described below, andfurther exemplified in the Examples section herein after.

By way of example, in one embodiment, a recombinant microorganism of theinvention is produced by a method comprises the following steps:

-   a. introduction into a shuttle microorganism of (i) of an expression    construct/vector as described herein and (ii) a methylation    construct/vector comprising a methyltransferase gene; expression of    the methyltransferase gene;-   b. isolation of one or more constructs/vectors from the shuttle    microorganism; and,-   c. introduction of the one or more construct/vector into a    destination microorganism.

In one embodiment, the methyltransferase gene of step B is expressedconstitutively. In another embodiment, expression of themethyltransferase gene of step B is induced.

The shuttle microorganism is a microorganism, preferably a restrictionnegative microorganism, that facilitates the methylation of the nucleicacid sequences that make up the expression construct/vector. In aparticular embodiment, the shuttle microorganism is a restrictionnegative E. coli, Bacillus subtillis, or Lactococcus lactis.

The methylation construct/vector comprises a nucleic acid sequenceencoding a methyltransferase.

Once the expression construct/vector and the methylationconstruct/vector are introduced into the shuttle microorganism, themethyltransferase gene present on the methylation construct/vector isinduced. Induction may be by any suitable promoter system although inone particular embodiment of the invention, the methylationconstruct/vector comprises an inducible lac promoter and is induced byaddition of lactose or an analogue thereof, more preferablyisopropyl-β-D-thio-galactoside (IPTG). Other suitable promoters includethe ara, tet, or T7 system. In a further embodiment of the invention,the methylation construct/vector promoter is a constitutive promoter.

In a particular embodiment, the methylation construct/vector has anorigin of replication specific to the identity of the shuttlemicroorganism so that any genes present on the methylationconstruct/vector are expressed in the shuttle microorganism. Preferably,the expression construct/vector has an origin of replication specific tothe identity of the destination microorganism so that any genes presenton the expression construct/vector are expressed in the destinationmicroorganism.

Expression of the methyltransferase enzyme results in methylation of thegenes present on the expression construct/vector. The expressionconstruct/vector may then be isolated from the shuttle microorganismaccording to any one of a number of known methods. By way of exampleonly, the methodology described in the Examples section describedhereinafter may be used to isolate the expression construct/vector.

In one particular embodiment, both construct/vector are concurrentlyisolated.

The expression construct/vector may be introduced into the destinationmicroorganism using any number of known methods. However, by way ofexample, the methodology described in the Examples section hereinaftermay be used. Since the expression construct/vector is methylated, thenucleic acid sequences present on the expression construct/vector areable to be incorporated into the destination microorganism andsuccessfully expressed.

It is envisaged that a methyltransferase gene may be introduced into ashuttle microorganism and over-expressed. Thus, in one embodiment, theresulting methyltransferase enzyme may be collected using known methodsand used in vitro to methylate an expression plasmid. The expressionconstruct/vector may then be introduced into the destinationmicroorganism for expression. In another embodiment, themethyltransferase gene is introduced into the genome of the shuttlemicroorganism followed by introduction of the expressionconstruct/vector into the shuttle microorganism, isolation of one ormore constructs/vectors from the shuttle microorganism and thenintroduction of the expression construct/vector into the destinationmicroorganism.

It is envisaged that the expression construct/vector and the methylationconstruct/vector as defined above may be combined to provide acomposition of matter. Such a composition has particular utility incircumventing restriction barrier mechanisms to produce the recombinantmicroorganisms of the invention.

In one particular embodiment, the expression construct/vector and/or themethylation construct/vector are plasmids.

Persons of ordinary skill in the art will appreciate a number ofsuitable methyltransferases of use in producing the microorganisms ofthe invention. However, by way of example the Bacillus subtilis phageΦT1 methyltransferase and a methyltransferase having the sequence of SEQID 14 or a functionally equivalent variant thereof may be used. Nucleicacids encoding suitable methyltransferases will be readily appreciatedhaving regard to the sequence of the desired methyltransferase and thegenetic code. In one embodiment, the nucleic acid encoding amethyltransferase is as for SEQ ID 15 or it is a functionally equivalentvariant thereof.

Any number of constructs/vectors adapted to allow expression of amethyltransferase gene may be used to generate the methylationconstruct/vector. However, by way of example, the plasmid described inthe Examples section hereinafter may be used.

Methods of Production

The invention provides a method for the production of one or moredesirable products by microbial fermentation of a substrate using arecombinant microorganism of the invention.

Any substrate which is appropriate for anaerobic fermentation may beused for the fermentation, including, for example, carbohydrates,sugars, substrates comprising CO, substrates comprising carbon dioxideand hydrogen, glycerol, fatty acids, starch, molasses, pentoses andhexoses sugars, biomass. In one embodiment, the substrate is a substratecomprising CO. In this embodiment, the methods of the invention may beused to reduce the total atmospheric carbon emissions from an industrialprocess.

Preferably, the fermentation comprises the steps of fermenting asubstrate in a bioreactor to produce the one or more products using arecombinant microorganism of the invention.

In one embodiment the method comprises the steps of:

-   -   (a) providing a substrate to a bioreactor containing a culture        of one or more microorganism of the invention; and    -   (b) anaerobic fermentation of the culture in the bioreactor to        produce the one or more products.

In one embodiment the method comprises the steps of:

-   -   (a) capturing CO-containing gas produced as a result of the        industrial process, before the gas is released into the        atmosphere;    -   (b)the anaerobic fermentation of the CO-containing gas to        produce the one or more products by a culture containing one or        more microorganism of the invention.

In an embodiment of the invention, the gaseous substrate fermented bythe microorganism is a gaseous substrate containing CO. The gaseoussubstrate may be a CO-containing waste gas obtained as a by-product ofan industrial process, or from some other source such as from automobileexhaust fumes. In certain embodiments, the industrial process isselected from the group consisting of ferrous metal productsmanufacturing, such as a steel mill, non-ferrous products manufacturing,petroleum refining processes, gasification of coal, electric powerproduction, carbon black production, ammonia production, methanolproduction and coke manufacturing. In these embodiments, theCO-containing gas may be captured from the industrial process before itis emitted into the atmosphere, using any convenient method. The CO maybe a component of syngas (gas comprising carbon monoxide and hydrogen).The CO produced from industrial processes is normally flared off toproduce CO₂ and therefore the invention has particular utility inreducing CO₂ greenhouse gas emissions and producing butanol for use as abiofuel. Depending on the composition of the gaseous CO-containingsubstrate, it may also be desirable to treat it to remove any undesiredimpurities, such as dust particles before introducing it to thefermentation. For example, the gaseous substrate may be filtered orscrubbed using known methods.

It will be appreciated that for growth of the bacteria and substrate tothe one or more product(s) to occur, in addition to the substrate, asuitable liquid nutrient medium will need to be fed to the bioreactor.The substrate and media may be fed to the bioreactor in a continuous,batch or batch fed fashion. A nutrient medium will contain a number ofcompounds sufficient to permit survival and/or growth of themicro-organism used, as known in the art. Suitable anaerobic and aerobicfermentation media are known in the art. For example, suitable media aredescribed Biebel (2001). However, the present invention offers theadvantage of not having to include in the media one or more vitamin, asthe recombinant microorganism is able to produce it. Accordingly,minimal media may be used, reducing costs. In addition, growth andfermentation of recombinant microorganisms typically involves theaddition to the media of a selection compound, typically one or moreantibiotic, so that the recombinant microorganism is selected for andany contaminating microorganisms do not survive. The use of antibioticsincreases the cost of fermentation, and there may be other downsidessuch as toxicity. The present invention obviates the need forantibiotics. In one embodiment of the invention the media is asdescribed in the Examples section herein after.

The fermentation should desirably be carried out under appropriateconditions for the substrate-to-the one or more product(s) fermentationto occur. Reaction conditions that should be considered includepressure, temperature, gas flow rate, liquid flow rate, media pH, mediaredox potential, agitation rate (if using a continuous stirred tankreactor), inoculum level, maximum substrate concentrations to ensurethat it does not become limiting, and maximum product concentrations toavoid product inhibition.

Where a substrate comprising CO is used, it is often desirable toincrease the CO concentration of the substrate stream (or CO partialpressure in a gaseous substrate) and thus increase the efficiency offermentation reactions where CO is a substrate. Operating at increasedpressures allows a significant increase in the rate of CO transfer fromthe gas phase to the liquid phase where it can be taken up by themicro-organism as a carbon source for the production of the one or moreproducts. This in turn means that the retention time (defined as theliquid volume in the bioreactor divided by the input gas flow rate) canbe reduced when bioreactors are maintained at elevated pressure ratherthan atmospheric pressure. The optimum reaction conditions will dependpartly on the particular micro-organism of the invention used. However,in general, it is preferred that the fermentation be performed atpressure higher than ambient pressure. Also, since a given CO-to-the oneor more product(s) conversion rate is in part a function of thesubstrate retention time, and achieving a desired retention time in turndictates the required volume of a bioreactor, the use of pressurizedsystems can greatly reduce the volume of the bioreactor required, andconsequently the capital cost of the fermentation equipment. Accordingto examples given in U.S. Pat. No. 5,593,886, reactor volume can bereduced in linear proportion to increases in reactor operating pressure,i.e. bioreactors operated at 10 atmospheres of pressure need only be onetenth the volume of those operated at 1 atmosphere of pressure.

By way of example, the benefits of conducting a gas-to-ethanolfermentation at elevated pressures has been described. For example, WO02/08438 describes gas-to-ethanol fermentations performed underpressures of 30 psig and 75 psig, giving ethanol productivities of 150g/l/day and 369 g/l/day respectively. However, example fermentationsperformed using similar media and input gas compositions at atmosphericpressure were found to produce between 10 and 20 times less ethanol perlitre per day.

It is also desirable that the rate of introduction of the CO-containinggaseous substrate is such as to ensure that the concentration of CO inthe liquid phase does not become limiting. This is because a consequenceof CO-limited conditions may be that the product is consumed by theculture.

The composition of gas streams used to feed a fermentation reaction canhave a significant impact on the efficiency and/or costs of thatreaction. For example, O2 may reduce the efficiency of an anaerobicfermentation process. Processing of unwanted or unnecessary gases instages of a fermentation process before or after fermentation canincrease the burden on such stages (e.g. where the gas stream iscompressed before entering a bioreactor, unnecessary energy may be usedto compress gases that are not needed in the fermentation). Accordingly,it may be desirable to treat substrate streams, particularly substratestreams derived from industrial sources, to remove unwanted componentsand increase the concentration of desirable components.

In certain embodiments a culture of a bacterium of the invention ismaintained in an aqueous culture medium (which does not include one ormore vitamins in accordance with the invention). Preferably the aqueousculture medium is a minimal microbial growth medium. Suitable media areknown in the art and described for example in U.S. Pat. Nos. 5,173,429and 5,593,886 and WO 02/08438, and as described in the Examples sectionherein after.

The one or more products may be recovered from the fermentation broth bymethods known in the art, such as fractional distillation orevaporation, pervaporation, gas stripping and extractive fermentation,including for example, liquid-liquid extraction.

In certain preferred embodiments of the invention, the one or moreproducts are recovered from the fermentation broth by continuouslyremoving a portion of the broth from the bioreactor, separatingmicrobial cells from the broth (conveniently by filtration), andrecovering one or more products from the broth. Alcohols mayconveniently be recovered for example by distillation. Acetone may berecovered for example by distillation. Any acids produced may berecovered for example by adsorption on activated charcoal. The separatedmicrobial cells are preferably returned to the fermentation bioreactor.The cell free permeate remaining after any alcohol(s) and acid(s) havebeen removed is also preferably returned to the fermentation bioreactor.

Vitamins may be recovered using any appropriate means. However, by wayof example they may be recovered by concentration and drying of thecells (e.g. centrifugation or spray drying) with subsequent extraction(e.g. in alcohol with purification and filtration using chromatographyor simply by differential centrifugation) and crystallization (Survaseet al., 2006, Food Technol. Biotechnol. 44: 381-96).

In one embodiment, the one or more product (in one particular embodimentone or more vitamin) is recovered from the fermentation and passed toone or more further bioreactor to support the growth of, or support thefermentation of a substrate by, one or more second microorganism.

In one embodiment, the one or more second microorganism is amicroorganism which is unable to produce, or unable to produce atsufficient levels, of the one or more vitamin(s) and requires its growthor fermentation media to be supplemented with the one or more vitamin(s)to ensure or maintain growth or fermentation, or to increase theefficiency of growth or fermentation.

The one or more product recovered from the first fermentation may bepassed to one or more further bioreactor using any suitable conduit.

In one embodiment, the methods of the invention may further comprisefermentation of a substrate by the one or more second microorganism toproduce one or more products. In one embodiment, the one or moreproducts may then be recovered from the fermentation broth.

Methods of Selection

The invention also provides a method for the selection of amicroorganism A in a mixed population of microorganisms, whereinmicroorganism A is a recombinant microorganism comprising at least oneexogenous nucleic acid which is adapted to express one or more enzymesin one or more vitamin biosynthesis pathway which produces one or morevitamin(s) which is needed for the growth of the mixed population ofmicroorganisms.

The method comprises subjecting the mixed population of microorganismsto culture conditions including a media which lacks the one or morevitamin(s) which is needed for the growth of the microorganisms. Thosemicroorganisms not able to produce the one or more vitamin(s), will notgrow or will be selected against.

In other embodiments, the one or more of the enzyme is as herein beforedescribed. The one or more vitamin is as herein before described. In oneparticular embodiment, the one or more vitamin is chosen from thiamine,pathothenate, riboflavin, nicotinic acid, pyridoxine, biotin, folicacid, and/or cyanocobalamine.

The “culture conditions” may be any suitable conditions which allow forat least the maintenance of a culture of microorganism A and includeconditions suitable for growth and/or fermentation. Skilled persons willappreciate suitable conditions, having regard to the nature of themicroorganism, and the information contained herein. However, by way ofexample, growth conditions include suitable environmental conditionsincluding pH, presence or absence of oxygen and other gases, salinity,temperature and the like.

Any suitable media may be used, provided it lacks the one or morevitamin needed for the growth of the microorganisms (which can beproduced by microorganism A) as described herein before. Skilled personswill readily appreciate a variety of appropriate media. However, in oneembodiment, the media is a minimal media.

While the invention overcomes the need to use alternative selectionmeans or supplement media with ingredients such as antibiotics, thesemethods could be combined with the current invention if desired.

The method of this aspect of the invention may be useful to distinguishbetween recombinant and non-recombinant microorganisms during theprocess of producing recombinant microorganisms—for example,distinguishing successfully transformed bacteria during a transformationprocess.

The method may also be useful for the purpose of selecting againstcontaminating microorganisms during laboratory or commercial scaleculturing of microorganisms and/or fermentation reactions. Withoutselection measures, undesirable microorganisms may grow to the detrimentof a desired microorganism, or otherwise affect the efficiency of theculture or fermentation reaction.

Accordingly, the invention also provides a means of preventing thegrowth of one or more undesirable microorganism in a microbial cultureor a fermentation broth, wherein the microbial culture or fermentationbroth comprises microorganism A and a nutrient media, whereinmicroorganism A is a recombinant microorganism comprising at least oneexogenous nucleic acid which is adapted to express one or more enzymesin one or more vitamin biosynthesis pathway which produces one or morevitamin(s) which is needed for the growth of microorganism A and theundesirable microorganism(s) such that the microorganism A can producethe one or more vitamin(s), wherein the media lacks the one or morevitamin(s).

The invention further provides a method for the selective growth orculture of a microorganism A, and wherein microorganism A is arecombinant microorganism comprising at least one exogenous nucleic acidwhich is adapted to express one or more enzymes in one or more vitaminbiosynthesis pathway which produces one or more vitamin(s) which areneeded for the growth of the microorganism, such that the microorganismA can produce the one or more vitamin(s), and wherein the growth orculture media lacks the one or more vitamin(s).

In one embodiment, the conditions select against the growth of one ormore undesirable microorganism(s).

In another aspect, the invention provides a method for the production ofone or more products by microbial fermentation of a substrate by amicroorganism A, wherein microorganism A is a recombinant microorganismcomprising at least one exogenous nucleic acid which is adapted toexpress one or more enzymes in one or more vitamin biosynthesis pathwaywhich produces one or more vitamin(s) which are needed for the growth ofthe microorganism, such that the microorganism A can produce the one ormore vitamin(s), and wherein fermentation occurs in or on a growth mediawhich lacks the one or more vitamin(s).

In one embodiment, the conditions select for growth of microorganism Aand against the growth of one or more undesirable microorganism(s).

It should be appreciated that the microorganisms, including recombinantmicroorganism A, of this aspect of the invention may be chosen from anymicroorganism of interest, and are not limited to anaerobes. However, inone embodiment they are chosen from the group of anaerobicmicroorganisms. In one embodiment, they are chosen from the group ofcarboxydotrophic acetogens.

The methods described herein before to produce the anaerobic recombinantmicroorganisms of other aspects of the invention, as well as methodsknown in the art, may be readily employed to generate the recombinantmicroorganisms to be selected in this aspect of the invention, adjustedto suit a particular microorganism where necessary. For example, wherethe microorganism is not an anaerobe, aerobic conditions can be used.The parental microorganism may be chose from any class ofmicroorganisms, and are not limited to anaerobes. However, in oneembodiment they may be chosen from the group of anaerobic microorganismsand in one particular embodiment carboxydotrophic acetogens.

Similarly, the methods for growth and fermention described for otheraspects of the invention may be employed in this aspect of theinvention, with conditions adjusted, as necessary for the type ofmicroorganism of interest; for example, substituting anaerobic andaerobic conditions.

The invention also provides microorganisms cultured or grown inaccordance with a method herein before described, and products producedby a method as herein before described.

EXAMPLES

The invention will now be described in more detail with reference to thefollowing non-limiting examples.

Methods

Analysis of Vitamin Biosynthesis Pathways of Clostridium ljungdahlii, C.autoethanogenum and C. ragsdalei

The inventors analysed the genomes of the carboxydotrophic acetogensClostridium ljungdahlii (NC_(—)014328.1; Köpke et al., 2010, Proc. Nat.Acad. Sci. U.S.A., 107: 13087-13092), C. autoethanogenum and C.ragsdalei. It was found that both C. ljungdahlii, as well as C.autoethanogenum are unable to synthesize thiamine due to the lack of thethiamine biosynthesis protein ThiC that participates in the formation of4-Amino-5-hydroxymethyl-2-methylpyrimidine from1-(5′-Phosphoribosyl)-5-aminoimidazole ribonucleotide (AIR). ThiC is theonly required gene product that has been identified for the pyrimidinebiosynthesis in E. coli, S. typhimurium, and B. subtilis (Begeley et al,1999, Arch Microbiol, 171: 293-300). On the other hand, ThiC and thefull thiamine biosynthetsis pathway is present in C. ragsdalei and otherorganisms. Thiamine auxotrophy of C. ljungdahlii and C. autoethanogenumwas demonstrated in serum bottle and fermentation experiments,confirming that thiamine needs to be added in fermentation medium (seebelow).

The panthothenate/CoA biosynthesis pathway was found to be incomplete inall three organisms, Clostridium ljungdahlii, C. autoethanogenum and C.ragsdalei due to the lack of biosynthetic genes panBCD, encoding a3-methyl-2-oxobutanoate hydroxymethyltransferase (EC:2.1.2.11;catalyzing conversion of 5,10-Methylenetetrahydrofolate and3-Methyl-2-oxobutanoic acid to Tetrahydrofolate and 2-Dehydropantoate),pantoate-beta-alanine ligase (EC:6.3.2.1; catalyzing the reaction of(R)-Pantoate+beta-Alanine to Diphosphate+Pantothenate), and aspartate1-decarboxylase (EC:4.1.1.11; catalyzing the conversion of L-Aspartateto beta-Alanine)

This concept can be extended to other species of Clostridium, includingnon acetogenic Clostridial species or to other acetogenic species.Genomes can be obtained from public resources as NCBI (/genome/browse/)or KEGG (genome.jp/kegg-bin/get_htext) and then analysed for presence ofgenes encoding vitamin biosynthesis proteins listed in tables 1-8. Forexample, it was found that the genes panCD are also missing in anothercarboxydotrophic acetogen Acetobacterium woodii that has been recentlysequenced (Poehlein et al., 2012) or that panBCD genes are missing inanother Clostridia species as C. phytofermentans while all other genesof the panthothenate pathway are present.

Growth Experiments with Clostridium ljungdahlii, C. autoethanogenum andC. ragsdalei in Media without Thiamine

Experiments were performed using C. autoethanogenum DSM10061 andDSM23693 (a derivate of DSM10061) and C. ljungdahlii obtained from DSMZ(The German Collection of Microorganisms and Cell Cultures,Inhoffenstraβe 7 B, 38124 Braunschweig, Germany). C. ragsdalei ATCCBAA-622 was sourced from ATCC (American Type Culture Collection,Manassas, Va. 20108, USA).

All strains were cultivated at 37 C in chemically defined PETC mediawithout yeast extract (Table 9) using strictly anaerobic conditions andtechniques (Hungate, 1969, Methods in Microbiology, vol. 3B. AcademicPress, New York: 117-132; Wolfe, 1971, Adv. Microb. Physiol., 6:107-146). 30 psi carbon monioxide containing steel mill waste gas(collected from New Zealand Steel site in Glenbrook, NZ; composition:44% CO, 32% N₂, 22% CO₂, 2% H₂) served as sole carbon and energy source.

TABLE 9 PETC medium Concentration per Media component 1.0 L of mediaNH₄Cl 1 g KCl 0.1 g MgSO₄•7H₂O 0.2 g NaCl 0.8 g KH₂PO₄ 0.1 g CaCl₂ 0.02g Trace metal solution 10 ml Wolfe's vitamin solution 10 ml minusThiamine Resazurin (2 g/L stock) 0.5 ml NaHCO₃ 2 g Reducing agent0.006-0.008% (v/v) Distilled water Up to 1 L, pH 5.5 (adjusted with HCl)Wolfe's vitamin solution minus Thiamine per L of Stock Biotin 2 mg Folicacid 2 mg Pyridoxine hydrochloride 10 mg Riboflavin 5 mg Nicotinic acid5 mg Calcium D-(+)-pantothenate 5 mg Vitamin B₁₂ 0.1 mg p-Aminobenzoicacid 5 mg Lipoic acid 5 mg Thiamine 5 mg Distilled water To 1 L Tracemetal solution per L of stock Nitrilotriacetic Acid 2 g MnSO₄•H₂O 1 gFe(SO₄)₂(NH₄)₂•6H₂O 0.8 g CoCl₂•6H₂O 0.2 g ZnSO₄•7H₂O 0.2 mg CuCl₂•2H₂O0.02 g NaMoO₄•2H₂O 0.02 g Na₂SeO₃ 0.02 g NiCl₂•6H₂O 0.02 g Na₂WO₄•2H₂O0.02 g Distilled water To 1 L Reducing agent stock per 100 mL of stockNaOH 0.9 g Cystein•HCl 4 g Na₂S 4 g Distilled water To 100 mL

Growth experiments were then carried out in PETC media omitting thiamine(or panthothenate) from Wolf's vitamin solution. While C. ragsdalei wasable to grow over multiple subcultures in absence of thiamine, C.autoethanogenum and C. ljungdahlii weren't able to grow for more than 2subculture step in absence of thiamine (or no subculture step, if thecell were washed before inoculation to remove residual thiamine),confirming the results from the genome analysis that those strains areauxotroph to thiamine.

For C. autoethanogenum DSM23693, also a bioreactor experiment wascarried out, using a defined medium containing per litre: MgCl, CaCl₂(0.5 mM), KCl (2 mM), H₃PO₄ (5 mM), Fe (100 μM), Ni, Zn (5 μM), Mn, B,W, Mo, Se (2 μM) was prepared for culture growth. The media wastransferred into the bioreactor and autoclaved at 121° C. for 45minutes. After autoclaving, the medium was supplemented with Wolfe'sB-Vitamin solution (see above), and reduced with 3 mM Cysteine-HCl. Toachieve anaerobic state, the reactor vessel was sparged with nitrogenthrough a 0.2 μm filter. Gas flow of carbon monioxide containing steelmill waste gas was initially set at 80 ml/min, increasing to 120 ml/minduring mid exponential phase, while the agitation was increased from 250rpm to 350. Na₂S was dosed into the bioreactor at 0.25 ml/hr. Once theOD600 reached 0.4, the bioreactor was switched to a continuous mode at arate of 1.0 ml/min (Dilution rate 0.96 d⁻¹). Thiamine was dosed into thereactor separately using a syringe pump. The thiamine pump was turnedoff for six days between day twenty and twenty six. During the period,thiamine feeding was stopped, the culture died and the biomass andgas-uptake dropped considerably. Re-starting the thiamine feeding,regenerated growth and biomass and gas uptake went back to the samelevel as before (FIG. 1), demonstrating that C. autoethanogenum isauxotroph to thiamine as expected in serum bottle experiments and fromgenome analysis.

Cloning of Thiamine Biosynthesis Gene thiC

The thiC gene of Clostridium ragsdalei was cloned into an appropriatevector for expression. Standard Recombinant DNA and molecular cloningtechniques were used in this invention (Sambrook et al, MolecularCloning: A laboratory Manual, Cold Spring Harbour Labrotary Press, ColdSpring Harbour, 1989; Ausubel et al, Current protocols in molecularbiology, John Wiley & Sons, Ltd., Hoboken, 1987).

For genomic DNA extraction of C. ragsdalei and C. autoethanogenum, a 100ml of an exponentially growing culture was harvested (4000×g, 15 min, 4°C.), washed with potassium phosphate buffer (10 mM, pH 7.5) andre-suspended in 1.9 ml STE buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA,200 mM sucrose). The cells were incubated for 30 minutes with 300 μL oflysozyme (100 000 U) at 37° C. The lysis step was followed by a 10minute incubation with 10% (w/v) SDS. The RNA was digested at roomtemperature by adding 240 μL of 0.5 M EDTA (pH 8.0), 20 μL of 1 MTris-HCl (pH 7.5), and 100 μL of RNase A. Then, 100 μl Proteinase K (0.5U) were added and proteolysis took place for 3 h at 37° C. Finally, 600μl of sodium perchlorate (5 M) were added, followed by aphenol-chloroform extraction and an isopropanol precipitation.

A region (SEQ ID NO: 1) of the C. ragsdalei genome containing the thiCgene (SEQ ID NO: 2), as well as adjacenat purF genes (SEQ ID NO: 4 and6) encoding a amidophosphoribosyl transferase (SEQ ID NO: 5 and 7) andpromoter region including regulatory thi box element (FIG. 2) usingoligonucleotides ThiC-ApaI-F (SEQ ID NO: 8:GCAGGGCCCAATACGATTATCTCCTTTC) and ThiC+PurF-Rev-SbfI (SEQ ID NO: 9:GCATCCTGCAGGTAAATTTTGTTCTTCATT) ordered from Life Technologies.Inclusion of the PurF may not be necessary, as both C. autoethanogenumand C. ljungdahlii contain a purF gene.

The amplification was performed on an Applied Biosystems GeneAmp PCRsystem 9700, using iProof HF DNA polymerase (Bio-Rad) and FailSafe 2×PCR premix E buffer (Epicentre). The genes were amplified using thefollowing program: Initial denaturation at 98° for two minutes, followedby thirty two cycles of denaturation (95° C. for 30 s), annealing (53°C. for 30 s), and extension (68° C. for 3 minutes). A final five minuteextension step at 72° C. completed the amplification of the 3063 byfragment.

Obtained PCR fragment was cloned into shuttle vector pMTL85246 (SEQ IDNO: 10) using ApaI and SbfI (New England Biolabs) to create plasmidpMTL85246-thiC-purF (SEQ ID NO: 11; FIG. 3). Vector pMTL85246 is aderivate of pMTL85240 with the C. autoethanogenumphosphotransacetylase-acetate kinase pta-ack operon promoter region (SEQID NO: 12) cloned in via NotI and NdeI.

The DNA fragments were incubated with ApaI and BSA at 25° C. for threehours, followed by SbfI addition and Incubation at 37° C. for a furtherthree hours. A double digest at 37 C was carried out for NotI and NdeI.All Enzymes were inactivated by twenty minute incubation at 65° C.Vector pMTL85240 and pMTL85246 was dephosphorylated using ShrimpAlkaline Phosphotase (Fermentas) at 37° C. for one hour. The purifiedDNA fragments were ligated with T4 DNA ligase (New England Biolabs). Thereaction was left to incubate at 16° C. overnight and the enzyme wasinactivated with ten minute incubation at 65° C.

5 μL of the ligation mixture was transformed into electro-competentcells of a thiC negative strain of E. coli JW3958-1 [thiC765(del)::kan(Baba et al, 2006, Mol. Syst. Biol., 2: 1-11)] obtained from the ColiGenetic Stock Centre (CGSC). After regeneration in LB media for 30 mins,cells were washed twice and selection was carried out using the thiCgene as selectable marker in M9 medium (Table 10) without thiamine.Colony formation took 2-3 days, and all screened colonies were positive,without any background growth. This is important when using asselectable marker and was surprisingly enabled by special treatment ofcells such as washing the cells after regeneration. When expressingplant thiC gene using an antibiotic selectable marker in E. coli,significant background growth was observed (Kong et al., 2008, CellResearch 18: 566-576).

Presence of the plasmid was confirmed using colony PCR (Intron iTaq PCRpremix). The PCR conditions were: 94° for three minutes, followed bythirty two cycles of denaturation (94° C. for 30 s), annealing (53° C.for 30 s), and extension (72° C. for 3 minutes). A final seven minuteextension step at 72° C. completed the amplification of the 3063 byfragment. The presence of the insert and plasmid was also confirmedusing restriction digestion with NdeI (NEB) giving two bands (4269 byand 2325 bp). The insert of the plasmid was fully sequenced to confirmsequence identity.

TABLE 10 M9 minimal media Media Concentration per component 1.0 L ofmedia M9 Salts 200 ml (see below) 1M MgSO₄ 2 ml 20% glucose 20 ml 1MCaCl₂ 100 μl Distilled water To 1 L M9 Salts per L of Stock Na₂HPO₄ 64 gKH₂PO₄ 15 g NaCl 2.5 g NH₄Cl 5 g Distilled water To 1 LUse of thiC as Selectable Marker in C. autoethanogenum

Prior to transformation in C. autoethangenum, plasmid DNA was in vivomethylated using E. coli strain XL1Blue MRF′ and methylation plasmidpGS20 (SEQ ID NO: 13) carrying a designed methyltransferase (SEQ ID NO:14) under control of an inducible lac promoter (SEQ ID NO: 15) asdescribed in WO 2012/053905. Methylated plasmid DNA was purified usingthe PureLink™ HiPure Plasmid Purification Kit (Life Technologies). Cellswere growing up in PET media (Table 9) with thiamine plus 1 g/L yeastextract and 5 g/L fructose plus steel-mill gas as carbon and energysource. A 50 mL culture of C. autoethanogenum DSM23693 was subculturedto fresh media for 3 consecutive days. These cells were used toinoculate 50 ml PETC media containing 40 mM DL-threonine at anOD_(600nm) of 0.05. When the culture reached an OD_(600nm) of 0.4, thecells were transferred into an anaerobic chamber and harvested at4,700×g and 4° C. The culture was twice washed with ice-coldelectroporation buffer (270 mM sucrose, 1 mM MgCl2, 7 mM sodiumphosphate, pH 7.4) and finally suspended in a volume of 600 μl freshelectroporation buffer. This mixture was transferred into a pre-cooledelectroporation cuvette with a 0.4 cm electrode gap containing 1 μg ofthe methylated plasmid mix and immediately pulsed using the Gene pulserXcell electroporation system (Bio-Rad) with the following settings: 2.5kV, 600Ω, and 25 μF. Time constants of 3.7-4.0 ms were achieved.Regeneration was carried out in 5 mL PETC media that has 10 g/L MES(2-(N-morpholino) ethanesulfonic acid) as buffer.

Cells were then either plated out on PETC-MES solid media (1.2% BactoAgar) with thiamine and yeast extract and also 5 μg/mL clarithromycin asselectable agent, or washed twice and plated on PETC-MES solid mediawithout yeast extract and without thiamine. In both cases, over 50positive colonies carrying the plasmid were obtained per plate, after 3days using clarithromycin as selectable agent and ermC as selectablemarker and after 6 days on plates without yeast extract and thiamineusing thiC as selectable marker.

Growth of C. autoethanogenum Engineered with thiC in Absence of Thiamine

Single colonies were picked and growth experiments were performed tocompare growth in PETC media without thiamine and yeast extract andsteel mill gas as sole energy and carbon source using C. autoethanogenumDSM23693 wild-type and strain carrying plasmid pMTL85246-thiC-purF.While growth of the wild-type ceased after two subculture steps (orwithin one subculture step if the cells were washed before inoculation),the strain carrying plasmid pMTL85246-thiC-purF was able to growsustainable for multiple subculturing steps (regardless if the cellshave been washed or not) (FIG. 5). Experiments were carried out intriplicates using in serum bottles and a volume of 50 mL. The presenceof plasmid was checked by PCR.

This demonstrates that the organism is able to synthesize thiamine byitself and thiC can be used as selectable marker in carboxydotrophicacetogen C. autoethanogenum.

Use of panBCD as Selectable Marker

The inventors have identified that C. autoethanogenus, C. ljungdahliiand C. ragsdalei have an incomplete panthothenate pathway lackingbiosynthetic genes panBCD, encoding a 3-methyl-2-oxobutanoatehydroxymethyltransferase (EC:2.1.2.11; catalyzing conversion of5,10-Methylenetetrahydrofolate and 3-Methyl-2-oxobutanoic acid toTetrahydrofolate and 2-Dehydropantoate), pantoate-beta-alanine ligase(EC:6.3.2.1; catalyzing the reaction of (R)-Pantoate+beta-Alanine toDiphosphate+Pantothenate), and aspartate 1-decarboxylase (EC:4.1.1.11;catalyzing the conversion of L-Aspartate to beta-Alanine) In otheracetogens such as Acetobacterium woodii, whose genome (Poehlein et al,2012, PLoS One 7: e33439), genes panB and panC were also found to beabsent, while the rest of the panthothenate biosynthesis pathway ispresent.

The same principle as for thiC and thiamine as selectable marker andagent may be applied for panBCD and panthothenate in these organisms.While in case of thiC only one gene is required, here three genes aremissing. However, all three genes have been found to be organized in onecluster in for example C. beijerinckii (FIG. 4). This cluster(NC_(—)009617.1, 3038300-3034200) including promoter regions and genespanB (Cbei_(—)2610; Gene ID: 5293811; YP_(—)001309722.1), panC(Cbei_(—)2609; Gene ID: 5293810; YP_(—)001309721.1), and panD(Cbei_(—)2608; Gene ID: 5293809; YP_(—)001309720.1) may be amplifiedfrom genomic DNA of C. beijerinckii by PCR and cloned into an expressionvector as described for the thiC gene from C. ragsdalei. This constructcould then be used in a similar fashion as described for the thiCconstruct by omitting panthothenate instead of thiamine for expressionin a organism lacking any of these genes, such as carboxydotrophicacetogens C. autoethanogenum, C. ljungdahlii, A. woodii, and C.ragsdalei or a panBCD negative strain of E. coli like E. coli JW0129-1(panC750(del)::kan), JW0130-1 (panB751(del)::kan), and JW0127-2(panD748(del)::kan) (Baba et al, 2006, Mol. Syst. Biol., 2: 1-11) whichcan be obtained from the Coli Genetic Stock Centre (CGSC).

Cloning of panBCD

panBCD genes were cloned into an expression vector using GeneArtSeamless Cloning and Assembly kit (Life Technologies). Large PCR primerscontaining a 20 by overhang homology to the desired vector weredesigned.

Primer sequence panBCD- AGGAAATGAACATGAAACATGTGAAAAATACAGTATTAAC83155-F1 TTTTAAACAAG (SEQ ID NO: 16) panBCD-GACGTCGACTCTAGAGGATCTTATTCATTTGATTCATAAT GeneD-R1TAGTTATTTCTTTTATTG (SEQ ID NO: 17)

The panBCD sequence was PCR amplified using iProof high fidelity DNApolymerase from C. beijerinckii. The protocol to amplify the 2813 byfragment was: Initialisation 30 s, Danturation 10 s, annealing 30 s,Extension 2 minutes, and a final extension step of 7 minutes.

The DNA was purified using DNA Clean and Concentrator −5 (ZymoResearch). pMTL vector 83155 carrying the catP antibiotic resistancemarker along with the Ppta promoter was digested using NdeI and BamHI(Fermentas) and purified using DNA Clean and Concentrator −5 (ZymoResearch). 100 ng of the digested vector was mixed with a 2:1 molarratio of insert, along with 5× reaction buffer and 10× Enzyme mix. Vthemixture was incubated at room temperature for 30 minutes and immediatelytransformed into One Shot TOP10 E. coli competent cells. 50 μL oftransformed E. coli was spread on LB agar plates containing 5 μg/mlchloramphenicol. Four colonies were screened using iNtron Maxime PCRPreMix i-MAX II (Tech Dragon Limited) from an overnight incubation

Primer sequence M13F TGTAAAACGACGGCCAGT (SEQ ID NO: 18) M13 RCAGGAAACAGCTATGACC (SEQ ID NO: 19)

The protocol to amplify the 3404 by fragment was: Initialisation 30 s,Danturation 10 s, annealing 30 s, Extension 2 minutes, and a finalextension step of 7 minutes.

The four plasmids were further checked by restriction digests using NotIand EcoRV (fermentas). Expected sizes from digestion: 2343 by and 5383bp, undigested plasmid: 7726 by

The invention has been described herein, with reference to certainpreferred embodiments, in order to enable the reader to practice theinvention without undue experimentation. However, a person havingordinary skill in the art will readily recognise that many of thecomponents and parameters may be varied or modified to a certain extentor substituted for known equivalents without departing from the scope ofthe invention. It should be appreciated that such modifications andequivalents are herein incorporated as if individually set forth.Titles, headings, or the like are provided to enhance the reader'scomprehension of this document, and should not be read as limiting thescope of the present invention.

The entire disclosures of all applications, patents and publications,cited above and below, if any, are hereby incorporated by reference.However, the reference to any applications, patents and publications inthis specification is not, and should not be taken as, an acknowledgmentor any form of suggestion that they constitute valid prior art or formpart of the common general knowledge in any country in the world.

Throughout this specification and any claims which follow, unless thecontext requires otherwise, the words “comprise”, “comprising” and thelike, are to be construed in an inclusive sense as opposed to anexclusive sense, that is to say, in the sense of “including, but notlimited to”.

We claim:
 1. A process for converting CO in a gaseous CO-containingsubstrate into higher molecular weight products, the process comprising:a) passing the gaseous CO-containing substrate to a bioreactorcontaining a culture of carboxydotrophic acetogenic bacteria in aculture medium such that the bacteria convert the CO to higher moleculeweight products, and b) recovering the higher molecular weight productsfrom the bioreactor, wherein the carboxydotrophic acetogenic bacteriaare genetically engineered to express an enzyme in a biosyntheticpathway of an essential nutrient that is absent from the culture medium,and wherein the carboxydotrophic acetogenic bacteria are prototrophicfor an essential nutrient selected from the group consisting ofthiamine, pantothenate, riboflavin, nicotinic acid, pyridoxine, biotin,folic acid, and cyanocobalamine, by virtue of an exogenous gene encodingthe enzyme.
 2. The process of claim 1 wherein the bacteria areprototrophic for thiamine and/or pantothenate by virtue of anheterologous thiC gene and/or an heterologous panBCD gene cluster.
 3. Anisolated, genetically engineered carboxydotrophic acetogenic bacteriumwhich is prototrophic for a vitamin selected from the group consistingof thiamine, pantothenate, riboflavin, nicotinic acid, pyridoxine,biotin, folic acid, and cyanocobalamine, by virtue of an expressedexogenous gene encoding an enzyme in a biosynthetic pathway that makesthe vitamin.
 4. The bacterium of claim 3 which is prototrophic forthiamine and/or pantothenate by virtue of an heterologous thiC geneand/or an heterologous panBCD gene cluster.
 5. The bacterium of claim 3which is selected from the group consisting of Clostridiumautoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei,Clostridium carboxidivorans, Clostridium drakei, Clostridiumscatologenes, Clostridium aceticum, Clostridium formicoaceticum,Clostridium magnum, Butyribacterium methylotrophicum, Acetobacteriumwoodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum,Moorella thermoacetica, Moorella thermautotrophica, Sporomusa ovata,Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacter pfennigii, andThermoanaerobacter kiuvi.
 6. The bacterium of claim 3 which is unable toconvert 4-aminoimidazole ribonucleotide to4-amino-5-hydroxymethyl-2-methylpyrimidine in the absence of saidheterologous thiC gene.
 7. The bacterium of claim 3 which is auxotrophicfor thiamine when cured of a plasmid.
 8. The bacterium of claim 3 whichis auxotrophic for pantothenate when cured of a plasmid.
 9. Thebacterium of claim 4 wherein the heterologous panBCD gene cluster isfrom C. beijerenckei.
 10. A method of culturing an isolated, geneticallyengineered carboxydotrophic acetogenic bacterium which is prototrophicfor a vitamin selected from the group consisting of thiamine,pantothenate, riboflavin, nicotinic acid, pyridoxine, biotin, folicacid, and cyanocobalamine, by virtue of an expressed exogenous geneencoding an enzyme in a biosynthetic pathway that makes the vitamin, themethod comprising, growing the bacterium in a medium comprising agaseous carbon source, wherein the carbon source comprises CO.
 11. Amethod of culturing an isolated, genetically engineered carboxydotrophicacetogenic bacterium which is prototrophic for a vitamin selected fromthe group consisting of thiamine, pantothenate, riboflavin, nicotinicacid, pyridoxine, biotin, folic acid, and cyanocobalamine, by virtue ofan expressed exogenous gene encoding an enzyme in a biosynthetic pathwaythat makes the vitamin, the method comprising, growing the bacterium ina medium comprising an energy source, wherein the energy sourcecomprises CO.
 12. The method of claim 10 wherein the bacterium comprisesan heterologous thiC gene and the medium is devoid of thiamine.
 13. Themethod of claim 11 wherein the bacterium comprises an heterologous thiCgene and the medium is devoid of thiamine.
 14. The method of claim 10wherein the bacterium comprises an heterologous panBCD gene cluster andthe medium is devoid of pantothenate.
 15. The method of claim 11 whereinthe bacterium comprises an heterologous panBCD gene cluster and themedium is devoid of pantothenate.
 16. The method of claim 10 wherein thegaseous carbon source comprises a product selected from the groupconsisting of automobile exhaust fumes, waste gas from ferrous metalproducts manufacturing, waste gas from non-ferrous productsmanufacturing, waste gas from petroleum refining processes, waste gasfrom gasification of coal, waste gas from electric power production,waste gas from carbon black production, waste gas from ammoniaproduction, waste gas from methanol production, waste gas from cokemanufacturing, and syngas.
 17. The method of claim 11 wherein thegaseous carbon source comprises a product selected from the groupconsisting of automobile exhaust fumes, waste gas from ferrous metalproducts manufacturing, waste gas from non-ferrous productsmanufacturing, waste gas from petroleum refining processes, waste gasfrom gasification of coal, waste gas from electric power production,waste gas from carbon black production, waste gas from ammoniaproduction, waste gas from methanol production, waste gas from cokemanufacturing, and syngas
 18. A method for transferring an heterologousnucleic acid into a population of carboxydotrophic acetogenic bacteriawhich are auxotrophic for an essential nutrient, the method comprising:a) transforming the bacteria with a first nucleic acid which comprisesan exogenous gene in a biosynthetic pathway of an essential nutrient,said gene operably linked to a promoter; and b) selecting for bacteriawhich are prototrophic for the essential nutrient among the transformedbacteria.
 19. The method of claim 18 wherein the step of transformingcomprises co-transforming the bacteria with a second nucleic acid whichcomprises an heterologous or endogenous gene conferring a desiredproperty when expressed in the bacterium.
 20. The method of claim 18wherein the essential nutrient is selected from the group consisting ofthiamine, pantothenate, riboflavin, nicotinic acid, pyridoxine, biotin,folic acid, and cyanocobalamine.
 21. The method of claim 20 wherein theessential nutrient is thiamine or pantothenate, and the heterologousnucleic acid is a thiC gene or a panBCD gene cluster, respectively. 22.The method of claim 18 further comprising the step of screeningprototrophic, transformed bacteria for the presence of the first nucleicacid or second nucleic acid.
 23. The method of claim 19 wherein thefirst and second nucleic acids are treated to form methylated first andsecond nucleic acids prior to the step of co-transforming.